CHRH antagonists with low histamine release

ABSTRACT

Antide is the decapeptide, N--Ac--D--2--Nal,D--pClPhe, D--3--Pal, Ser,NicLys, D--NicLys, Leu, ILys, Pro, D--Ala,NH 2  which is an antagonist of luteinizing hormone releasing hormone (LHRH). This decapeptide, like others of the present invention, has high antiovulatory activity (AOA) and releases negligible histamine. Antide is scheduled for scale-up, safety testing and evaluation in the experimental primate and in clinical medicine. Numerous other peptides having structures related to Antide were prepared and tested. These peptides had variations primarily in positions 5, 6, 7, and 8. Of these, N--Ac--D--2--Nal, D--pClPhe,D--3--Pal,Ser,PicLys,cis--DpzACAla, Leu,ILys,pro,D--Ala--NH 2  was one of the most potent and had higher antiovulatory activity than Antide, i.e. 73%/0.25 ug and 100%/0.5 ug vs. 36%/0.5 ug and 100%/1.0 ug. Antide showed significant, (p&lt;0.001) duration of action, when injected at a dose of 10 ug, 44 hours before 50 ng of the agonist, [D--3--Qal 6  ]--LHRH. Antide showed oral AOA at 600 ug (73%) and at 1200 ug (100%) with negligible difference being found between water and corn oil oral formulations.

This is a continuation-in-part of U.S. patent application Ser. No.088,431 filed Aug. 24, 1987 now U.S. Pat. No. 4,935,491, issued Jun. 19,1990 which is incorporated by reference herein. Benefit under 35 U.S.C.§120 is also claimed for this application, which is a continuationapplication of International Application No. PCT/US 88/02922 having theInternational Filing Date of Aug. 24, 1988.

Research related to the development of this invention was supported inpart by the Contraceptive Branch of the National Institutes of ChildHealth and Human Development, contract no. NOI HD-6-2938 and to theRobert A. Welch Foundation.

BACKGROUND OF THE INVENTION

The present invention involves the design, synthesis and use ofsynthetic analogs of the luteinizing hormone releasing hormone (LHRH).An important achievement involved synthesis of analogs which functionedas antagonists of LHRH, were adequately potent to inhibit ovulation andallowed the release of only negligible amounts of histamine. Since therewas no way of reliably forecasting the structure of an antagonist havinghigh potency and very low histamine release, it was necessary to explorediverse approaches to discover a combination of structural featureswhich would yield an antagonist of LHRH having high potency forovulation inhibition and very low activity for histamine release.

Various peptides such as substance P, vasoactive intestinal peptide,gastrin, somatostatin, as well as others, are well known to cause therelease of histamine from mast cells. These cells are in many tissues,such as skin, lung and mesentery, gingiva, etc. Most cells have granulescontaining histamine and other mediators of inflammation which can bereleased by peptides to cause capillary dilation and increased vascularpermeability. When it was noted that an antagonist of LHRH, for example[Ac--D--2--Nal², D--4--F--Phe², D--TrP³, D--Arg⁶ ]--LHRH, caused edemaof the face and extremities when it was administered to rats, itappeared likely that such antagonists, if administered to human subjectsas a contraceptive agent, would cause serious edema of the face andelsewhere in the human body. Such side effects would likely prevent theadministration of such antagonists to human subjects.

The histamine-containing leukocyte is a basophile which can also releasehistamine when stimulated by many of the same peptides mentioned above.Basophiles differ biochemically from mast cells and such differences mayallow for both predictable and unpredictable histamine release inresponse to antagonists of LHRH. An antagonist of LHRH, to be usedclinically to prevent ovulation, should not significantly releaseamounts of histamine from either mast cells or basophiles.

The discovery of the side effects such as the edematogenic andanaphylactoid actions of LHRH antagonists made desireable the discoveryof new LHRH antagonists which prevented ovulation but did not releasesignificant histamine. These undesireable side effects have beenobserved in rats, and it is likely that the Food and Drug Administrationwould not allow the testing of such antagonists in human subjects.

Karten et al. (4), have reviewed available knowledge on the structuralcharacteristics for potent histamine release by antagonists of LHRH.Some of the most important findings are as follows. A most potent LHRHantagonist in triggering histamine release in vitro involved acombination of strongly basic D-amino acid side chains (Arg or Lys) atposition 6 and in close proximity to Arg⁸, and a cluster of hydrophobicaromatic amino acids at the N-terminus. Thus, there is no specific aminoacid of the ten amino acids which is solely responsible for histaminerelease. On the contrary, structural features ranging from theN-terminus (the amino acids in the first few positions, 1-4, etc.), andbasic amino acids toward the C-terminus (positions 6 and 8) somehowparticipate in histamine release. Even D--Ala in position 10 has someinfluence on histamine release, the rationale for which is unclear. Bythemselves, two basic side chains in close proximity, as in positions 6and 8, are insufficient alone to impart high release of histamine. Thecluster of hydrophobic amino acids at the N-terminus is insufficientalone for high histamine releasing activity. Even a hexapeptide fragmenthas revealed moderate histamine releasing potency. There seems to be nocorrelation between antiovulatory potency and histamine release of theseantagonists, in vitro.

In perspective, much of the entire chain of such decapeptide antagonistsmay have influence on histamine release. The same perspective appears tobe true, but to different degrees, for high antiovulatory activity.These LHRH antagonists are usually decapeptides which indicates thatthere are ten variables to adjust for a desired anti-ovulatory activityand ten variables to adjust for eliminating histamine releasingactivity. There are even further variations for each of these twentyvariables, the number of possible peptides to design, synthesize andassay becoming incalculable. Presumably, some of the ten variables maybe independent for anti-ovulatory activity and histamine releasingactivity while some variables may overlap for these two biologicalactivities. This situations presents a extraordinary difficulties tosolve before an antagonist of high potency for anti-ovulation and verylow potency for histamine release could be produced.

Diverse structural changes and combinations of the ten amino acidsfollowed by assays of both anti-ovulation and histamine releaseactivities should be performed in the hope that a potent antagonistessentially free of side effects would be discovered. The synthesis ofnew amino acids to introduce into the decapeptide chains should also beexplored since the commonly available amino acids might not suffice.

SUMMARY OF THE INVENTION

In the antagonists prepared according to the present invention, arginineand its derivatives were not utilized. Lysine was converted intoderivatives with acyl groups or with alkyl groups on the E-amino group.The amino acid ornithine was acylated or alkylated on the d-amino group.Both the L- and D- forms of lysine and the L-form of ornithine were usedin synthesizing these acyl and alkyl derivatives. Structurally relatedintermediates were also synthesized. All together, many new peptideswere synthesized by the basic and minimal concepts of ten variables foranti-ovulation activity and ten variables for histamine release, whichmay be independent or partially overlapping. On such a basis, the numberof such peptides that can be designed becomes overwhelming, and everyreasonable priority must be considered to reduce the number of peptidesto be synthesized in the hope that a discovery will be realized.

Certain peptides were synthesized, tested and found to demonstrateadvantageous peptides. Among these desireable peptides were thefollowing two. [N--Ac--D--2-Nal¹, D--pClPhe²,D--3--Pal³, NicLys⁵,D--NicLys⁶, ILys⁸, D--Ala¹⁰ ]-LHRH was effective to prevent ovulationand released remarkably little histamine. [N--Ac--D--2--Nal¹,D--pClPhe²,D--3--Pal³, PicLys⁵, D--PicLys⁶, ILys⁸, D--Ala¹⁰ ]-LHRH was twice aseffective as the above peptide, and released no more histamine than do"super agonists" of LHRH, which are presently being marketed by severalpharmaceutical companies.

These two new peptides, and yet additional related peptides describedherein provide acceptable balances of high anti-ovulatory activity andlow histamine release for full potential clinical utility.

The present invention involves the preparation and use of decapeptideshaving antiovulatory activity and with minimal histamine-releasingeffects. These decapeptides includes those comprising:

Ser⁴, PicLys⁵ and D-PicLys6;

N--Ac--D--2--Nal¹, D--pClPhe², Ser⁴, D--PicLys⁶ and Pro⁹ ;

N--Ac--D--2-Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, D--PicLys⁶, Pro⁹ andD--Ala¹⁰ ;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, NicLys⁵, Pro⁹ andD--Ala¹⁰ ;

N--Ac--D--2-Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, Leu⁷, Pro⁹ and D--Ala¹⁰;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, Leu⁷, Pro⁹ and D--Ser¹⁰;

D--pClPhe², Pro⁹ and D--Ala¹⁰ ;

D--pClPhe², Pro⁹ and Ser¹⁰ ;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, NicLys⁵, D--NicLys⁶, ILys⁸and D--Ala¹⁰ ;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, NicLys⁵, D--NicLys⁶, ILys⁸and D--Ala¹⁰ ;

N--Ac--D--2-Nal¹, D--pClPhe², D--3--Pal³, PicLys⁵, D--PicLys⁶, ILYs⁸ andD--Ala¹⁰ ;

N--Ac--D--2-Nal¹, D--pClPhe², D--3--Pal³, NicLys⁵, D--NicLys⁶, IOrn⁸ andD--Ala10;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, PicLys⁵, D--PicLys⁶, IOrn⁸and D--Ala¹⁰ ;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, MNicLys⁵, D--MNicLys⁶, IOrn⁸and D-Ala¹⁰ ;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, PzcLys⁵, D--PzcLys⁶, IOrn⁸and D--Ala¹⁰ ;

N--Ac--D--pClPhe¹ ; D--3--Pal³, Tyr⁵, D-NicLys⁶ and ILys⁸ ;

N--Ac--D--Cl₂ Phe¹, D--3--Pal³, Tyr⁵, D--NicLys⁶ and ILys⁸ ;

acylated Lys⁵, D-acylated Lys⁶ and N-alkylated diamino acid⁸ ;

NicLys⁵, D--NicLys⁶ and ILys⁸ ;

PicLys⁵, D--PicLys⁶ and ILys⁸ ;

NicLys⁵, D--NicLys⁶ and IOrn⁸ ;

PicLys⁵, D--PicLys⁶ and IOrn⁸ ;

MNicLys⁵, D--MNicLys⁶ and IOrn⁸ ;

PzcLys⁵, D--PzcLys⁶ and IOrn⁸ ;

Tyr⁵, D--NicLys⁶ and ILys⁸ ;

Tyr⁵, D--NicLys⁶ and IOrn⁸ ;

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, NicLys⁵, D--NicLys⁶,Leu⁷, ILys⁸, Pro⁹ and D--Ala¹⁰ NH₂ ; and

N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, PicLys⁵, cisD--PzACAla⁶, Leu⁷, ILys⁸, Pro⁹ and D--Ala¹⁰ NH₂.

The present invention further involves use of the above decapeptides ina process for inhibiting ovulation in an animal. This process comprisesadministering to said animal a decapeptide preferably having thestructure: N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, Ser⁴, NicLys⁵,D--NicLys⁶, Leu⁷, ILys⁸, Pro⁹ and D--Ala¹⁰ NH₂. Likewise, the inventiveprocess may be used to inhibit ovulation in an animal; to inhibit theonset of puberty in an animal; to inhibit the sexual impetus of ananimal; to alter the gonadal function of an animal; to inhibit thegrowth of hormone-dependent tumors in an animal; and to lower LH and FSHlevels in serum of post-menopausal women. These and other related useswill be apparent to those skilled in the art upon examination of thisspecification.

DESCRIPTIONS OF PREFERRED EMBODIMENTS

Abbreviations and formulas used herein include the following:

    ______________________________________                                        a =       alpha                                                               BOC =     t-butoxycarbonyl                                                    Br--Z =   o-bromobenzyloxycarbonyl                                            nBuOAc =  n-butylacetate                                                      n-BuOH =  n-butanol                                                           c =       cis                                                                 CDCl.sub.3 =                                                                            deuterochloroform                                                   CHCl.sub.3 =                                                                            chloroform                                                          CH.sub.2 Cl.sub.2 =                                                                     dichloromethane                                                     CH.sub.3 CN =                                                                           acetonitrile                                                        Cl--Z =   o-chlorobenzyloxycarbonyl                                           d =       delta                                                               DCC =     dicyclohexylcarbodiimide                                            DIEA =    diisopropylethylamine                                               DMF =     dimethylformamide                                                   E =       epsilon                                                             Et =      ethyl                                                               EtOAc =   ethyl acetate                                                       EtOH =    ethanol                                                             Et.sub.2 O =                                                                            diethyl ether                                                       HF =      hydrogen fluoride                                                   HOAc =    acetic acid                                                         KH.sub.2 PO.sub.4 =                                                                     potassium dihydrogen phosphate                                      MeOH =    methanol                                                            MgSO.sub.4 =                                                                            magnesium sulfate                                                   NH.sub.4 OAc =                                                                          ammonium acetate                                                    iPrOH =   2-propanol                                                          py =      pyridine                                                            t =       trans                                                               TFA =     trifluoroacetic acid                                                THF =     tetrahydrofuran                                                     TOS =     p-toluensulfonyl                                                    u =       micro                                                               Z =       benzyloxycarbonyl                                                   Abu =     2-aminobutyric acid                                                 ACAla =   aminocyclohexylalanine                                              Aile =    alloisoleucine                                                      AnGlu =   4-(4-methoxyphenylcarbamoyl)-2-                                               aminobutyric acid                                                   BzLys =   N.sup.E -benzoyllysine                                              Cit =     citrulline                                                          Cl.sub.2 Phe =                                                                          3,4-dichlorophenylalanine                                           CypLys =  N.sup.E -cyclopentyllysine                                          DMGLys =  N.sup.E -(N,N-dimethylglycyl)lysine                                 Dpo =     N.sup.d -(4,6-dimethyl-2-pyrimidyl)                                           ornithine                                                           Et.sub.2 hArg =                                                                         N.sup.G,N.sup.G -diethylhomoarginine                                FPhe =    A-fluorophenylalanine                                               HOBLys =  N.sup.E -(4-hydroxybenzoyl)lysine                                   Ilys =    N.sup.E -isopropyllysine                                            INicLys = N.sup.E -isonicotinoyllysine                                        IOrn =    N.sup.d -isopropylornithine                                         Me.sub.3 Arg =                                                                          N.sup.G,N.sup.G,N.sup.G1 -trimethylarginine                         Me.sub.2 Lys =                                                                          N.sup.E,N.sup.E -dimethyllysine                                     MNicLys = N.sup.E -(6-methylnicotinoyl)lysine                                 MPicLys = N.sup.E -(6-methylpicolinoyl)lysine                                 NACAla =  3(4-nicotinoylaminocyclohexyl)alanine                               2-Nal =   3-(2-naphthyl)alanine                                               NicLys =  N.sup.E -nicotinoyllysine                                           NicOrn =  N.sup.d -nicotinoylornithine                                        Nle =     norleucine, 2-aminohexanoic acid                                    NMeLeu =  N-methylleucine                                                     Nval =    norvaline, 2-aminopentanoic acid                                    PACAla =  3(4-picolinoylaminocyclohexyl)alanine                               3-Pal =   3-(3-pyridyl)alanine                                                pClPhe =  3-(4-chloro)phenylalanine                                           PicLys =  N.sup.E -picolinoyllysine                                           Pip =     piperidine-2-carboxylic acid                                        PmcLys =  N.sup.E -(4-pyrimidinylcarbonyl)lysine                              PmACAla = 3[4(4-                                                                        pyrimidinylcarbonyl)aminocyclohexyl]alanine                         PzACAla = 3(4-                                                                          pyrazinylcarbonylaminocyclohexyl)alanine                            3-PzAla = 3-pyrazinylalanine                                                  PzcLys =  N.sup.E -pyrazinylcarbonyllysine                                    Sar =     N-methylglycine                                                     TinGly =  3-thienylglycine                                                    ______________________________________                                    

Most natural amino acids were obtained from Peninsula Laboratories, SanCarlos, Calif. The hydroxyl group of Ser was protected as the benzylether, the phenolic hydroxyl group of Tyr as the Br--Z derivative, andE-amino group of Lys as the Cl--Z derivative, the guanidino group of Argand the imidazole group of His as the TOS derivatives. The a-aminofunction was protected as the BOC derivative. BOC-Orn(Z) was obtainedfrom Sigma Chemical Co., St. Louis, Mo. BOC--D--2--Nal, BOC--D--3--Pal,BOC--D--Cl₂ Phe, BOC--pClPhe and BOC--ILys(Z) dicyclohexylamine saltwere provided by the Southwest Foundation for Biomedical Research, SanAntonio, Tex. The benzhydrylamine hydrochloride resin was obtained fromBeckman Bioproducts, Palo Alto, Calif. The nitrogen content was about0.65 mmoles/g. The CH₂ Cl₂ was distilled before use.

The present invention involves the design, synthesis and use of LHRHantagonists with high antiovulatory potency and diminished activity torelease histamine (1). These new antagonists feature for example,D--N^(E) --nicotinoyllysine (D--NicLys) in position 6 and N^(E)--isopropyllysine (ILys) in position 8. The solution of D--Arg⁶,particularly in combination with Arg⁸ and a cluster of hydrophobicaromatic amino acid residues at the N-terminal, have been implicated inthe release of histamine (2-4).

Other reductions of anaphylactoid activity were obtained by increasingthe distance between the positive charges in positions 6 and 8 by Arg⁵and by inclusion of a neutral residue in position 6 as in[N--Ac--D--2--Nal¹, D--pClPhe², D--3--Pal³, Arg⁵,D--4(p-methoxybenzoyl)-2-aminobutyric acid⁶, D--Ala¹⁰ ]--LHRH (2-Nalrepresents 3-(2-naphthyl) alanine; PClPhe represents3(4-chlorophenyl)alanine; 3-Pal represents 3(3-pyridyl)alanine) byRivier et al. (5) and [N--Ac--D--2--Nal¹, D--aMepClPhe², D--Trp³, Arg⁵,D--Tyr⁶, D--Ala¹⁰ ]--LHRH (aMepClPhe represents 2methyl-3(4-chlorophenyl)alanine) by Roeske et al. (6). Furthermodifications in position 6 are reductive alkylation of D--Lys⁶ byHocart et al. (7), incorporation of N,N-diethylhomoarginine by Nestor etal. (8). The cyclic analogs recently synthesized by Rivier et al. didnot show any lowering in histamine release compared to the linearcounterparts (10).

From the peptides of the present invention, two were initially selectedas models for further design. The peptide [N--Ac--D--2--Nal¹,D--pClPhe², D--3--Pal³, NicLys⁵, D--NicLys⁶, ILys⁸, D--Ala¹⁰ ]-LHRH(named Antide) had an impressive combination of potency and lowhistamine release; antiovulatory activity (AOA) was 100% at lug and 36%at 0.5 ug; ED₅₀ for histamine release, in vitro, was consistently above300 ug/ml as compared to about 0.17 for the standard analog[N--Ac--D--2--Nal¹, D--pFPhe², D--Trp³, D--Arg⁶ ]--LHRH (pFPherepresents 3(4-fluorophenyl)alanine) (5). Another analog was identicalto Antide except for PicLys⁵ and D--PicLys⁶ (PicLys representsN-picoloyllysine); 100% AOA at 0.5 ug and 40% at 0.25 ug; ED₅₀, 93±11.

Included herein are results from LHRH analogs with acylatedaminocyclohexylalanine residues in position 6, from analogs in whichLeu⁷ has been substituted with other neutral residues from a comparisonof ILys⁸ vs. IOrn⁸, and from tests on oral activity and duration ofantagonists activity when administered orally or parenterally (s.c.)

Melting points are uncorrected. NMR data are reported as d-valuesdownfield from TMS.

Before acylation, the Z and Cl--Z groups of Lys and Orn were cleaved byhydrogenolysis in MeOH in the presence of 10% Pd/C.

BOC--D--BzLys was synthesized by acylation of BOC--D--Lys with benzoylchloride as described for the L- isomer by Bernardi et al. (17).

BOC--DMG--Lys was prepared by acylation of BOC--Lys with chloracetylchloride using the same method and reacting the crude product from 10mmoles BOC--Lys in 10 ml THF with 10 ml 40% aq. dimethylamine. Thereaction mixture was stirred 15 minutes in ice bath and then 2.5 hoursat room temperature. After evaporation in vacuo the crude product wasdissolved in 10 ml H₂ O and applied on a Bio-Rad AG1-X8 column, acetateform, 1×25 cm. The column was first washed with 200 ml water and thenthe product was eluted with 6% HOAc and lyophilized several times toremove the HOAc. Yield 60-70%. Amorphous mass. R_(f) (n-BuOH:py:HOAc:H₂O=30:10:3:12)=0.27. Purity>95%. NMR (CDCl₃):1.45,s,9H,t-butoxy group;1.85-1.48, PG,14 m,6H,B,y,d,CH₂ groups; 2.6,s,6H,N(CH₃)₂ ; 3.25,m,2H,E--CH₂ ; 3.37,s,2H,N--CH₂ --CO; 4.15,m,1H,a--CH.

The other acylated Lys derivatives in the tables were prepared fromBOC-D or L-Lys and the corresponding p-nitrophenyl ester.

p-Nitrophenyl nicotinate. To 9.85 g, 80 mmoles, nicotinic acid and 13.35g, 96 =moles p-nitrophenol in 250 ml DMF was added 16.5 g, 80 mmoles DCCwith stirring in ice-bath. After 1 hour at 0° C. and 3 hours at roomtemperature the urea was filtered off and the product was precipitatedby the addition of an equal volume of water. Filtration, drying in vacuoand recrystallization from i-PrOH gave 11.22 g, 57% of white needles,m.p. 172.5°-173° C. (24)

p-nitrophenyl isonicotinate was prepared, in the same manner 12 g, 61%,m.p. 139°-141° C., m.p. 137°-139° C. (18)

Also p-nitrophenyl 6-methylnicotinate was prepared in the same way.Yield from 70 mmoles 6-methylnicotinic acid: 6.0 g, 33% afterrecrystallization from MeOH. M.p. 156°-157° C. R_(f) (2% MeOH inCHCl₃)=0.57 NMR (CDCl₃): 2.7,s,3H,CH₃ ; 7.36,d,1H,py H⁵ ;7.45,m,2H,Hadjacent to the oxygen in the phenyl ring; 8.34,m,3H,H adjacent to theNO₂ group in the phenyl ring overlapping with py H⁴ ; 9.27,d,1H,py H².

P-nitrophenyl picolinate. 4.92 g, 40 mmoles, picolinic acid and 5.84 g,42 =moles p-nitrophenol were suspended/dissolved in 200 ml CH₂ Cl₂. Then8.24 g 40 mmoles, DCC was added in 20 ml CH₂ Cl₂ with vigorous stirring.Stirring was continued in room temperature for 17 hours. Then themixture was filtered and the filter cake washed with 30-40 ml CH₂ Cl₂.The raw product was first treated with 100 ml Et₂ O with stirring inice-bath and filtered. Recrystallization from 250 ul iPrOH gave 6.24 g,63% product. M.p. 154°-6° C. (dec.). M.p. 145°-7° C. (18).

Pyrazinecarboxylic acid p-nitrophenylester. This compound was preparedusing the same method as the previous compound. From 40 mmolespyrazinecarboxylic acid and 44 mmoles p-nitrophenol was obtained 35.2Moles, 88%, ester. M.p. 180°-182° C. (dec.). R_(f) (CHCl₃:MeOH=49:1)=0.72. NMR (CDCl₃): 7.5,m and 8.37 m,2H each, hydrogensadjacent to the oxygen and nitro group respectively in the phenol ring;8.84,m, 1H,pyrazine H⁵ ; 8.9,d,1H,pyrazine H⁶ ; 9.48,d,1H,pyrazine H³.

BOC--NicLys. 2.5 g BOC--Lys (L or D) was suspended in 200 ml DMF withstirring. Then 1.1 equivalent of p-nitrophenyl nicotinate was added andthe mixture stirred at room temperature for 36 hours. The mixture wasthen filtered and the filtrate evaporated to dryness at reduced pressureto yield a yellow oil. The residue was stirred with 2×50 ul Et₂ O inice-bath. The first Et₂ O phase was decanted, the second was filteredoff. Recrystallization from EtOAc/hexanes gave 2.05 g product, 58%(L-form). M.p. 138° C., lit (17) 138°-141° C. L-form [a] ²⁰ _(D) =-2.91°(MeOH), D-form [a]²⁰ _(D) =3.35° (MeOH).

L- and D-BOC-INicLys were prepared similarly by acylating 10 mmoles L orD BOC-Lys with p-nitrophenyl isonicotinate in 100 ml DMF, 40 hours, roomtemperature. The crude product was partitioned between 120 ml EtOAc and50 ul H₂ O. The EtOAc phase was extracted with 2×50 ml H₂ O and 50 mlbrine. The original aqueous phase was back-extracted with 30 ml EtOAc.The combined EtOAc phases were then dried (MgSO₄) and evaporated and theresidue was treated with Et₂₀ and recrystallized as above to give 1.07g, BOC- L-INicLys, 30.5%. The yield for the D compound was 1.26 g, 36%.NMR (Acetone d₆): 1.4, s, 9H, t-butoxy group; 1.8-1.48, m, 6H, B, y, d,--CH₂ --; 3.4,, t,2H,E--CH₂ ; 4.13,m1H,a--CH; 7.77,m,2H,py H⁵ and H³ ;8.70,m,2H,py H² and H⁶.

L- and D-BOC-PicLys. 1.23 g, 5 mmoles, of L- or D-BOC-Lys was stirredwith 1.34 g, 5.5 =moles, p-nitrophenyl picolinate in 60 ml DMF for 16hours. After filtration and evaporation and product was purified bycolumn chromatography on silica gel on a 4.5×32 cm column and thesolvent system n-BuOH:py:HOAc:H₂ O=30:10:3:12. The product afterchromatography was dissolved in EtOAc and washed with H₂ O, brine, driedand evaporated in vacuo. The yields were usually 60-70%. NMR (CDCl₃):1.43,s,9H,t-butoxy group; 1.73-1.45,m,6H,B,y,d--CH₂ ; 3.47,m,2H,E--CH₂ ;4.32,m,1H,a--CH; 7.43,m,1H,py H⁵ ; 7.85,m,1H,py H⁴ ; 8.55,m,1H,py H³ ;8.55,m,1H,py H⁶.

L- and D-BOC-MNicLys. 10 mmoles BOC-Lys and 10.5 mmoles p-nitrophenyl6-methylnicotinate were allowed to react in 150 ml DMF in the usualmanner. After 27 hours filtration and evaporation yielded a yellow oil.Et₂ O treatment (2×50 ml) gave 3.3 g product which was recrystallizedfrom 50 ul 20% MeOH in EtOAc/hexane. Yield 2.87 g, 78.6% (L-form). R_(f)(n-BuOH:py:HOAc:H₂ O=32:10:3:12)=0.61. NMR(CDCl₃): 1.46,s,9H,t-butoxygroup; 1.9-1.5,m,6H,B,y,d--CH₂ ; 2.57,s,3H,py CH₃ ; 3.36,m,2H,E--CH₂ ;4.11,m,1H,a--CH; 7.22,d,1H,py H⁵ ; 8.08,m,1H,py H⁴ ; 8.95,broad s,1H,pyH².

L- and D-BOC-PzcLys. Using the method above was obtained from 7.7 mmolespyrazine carboxylic acid p-nitrophenyl ester and 7 mmoles BOC-Lys, L orD, in 100 ml DMF about 6 mmoles product after recrystallization fromiPrOH. R_(f) (n-BuOH:py:HOAc:H₂ O=30:10:3:12)=0.47. NMR (CDCl₃):1.45,s,9H,t-butoxy group; 1.9-1.48,m,6H,B,y,d--CH₂ --; 3.51,m,2H,E--CH₂; 4.29,m,1H,a--CH; 8.52,q, 1H,pyrazine H⁵ ; 8.77,d,1H,pyrazine H⁶ ;9.41,d,1H,pyrazine H³.

BOC-L-NicOrn. This compound was prepared the usual way by reacting 7mmoles p-nitrophenyl nicotinate with 5 mmoles BOC-Orn in 75 ml DMF for36 hours. Evaporation and recrystallization from EtOAc gave 3.5 mmoles,70%, NicOrn, m.p. 143°-144° C. R_(f) (n-BuOH:HOAc:H₂ O=4:1:2)=0.70.NMR(CDCl₃): 1.45,s,9H,t-butoxy group; 7.46,m, 1H,py H⁵ ; 8.27,m,1H,py H⁴; 8.69,m, 1H,py H⁶ ; 9.05,m,1H,py H².

BOC-D-trans-NACAla. 1.43 g, 5 mmoles, BOC-D-trans-3(4-aminocyclohexyl)alanine (provided by the Southwest Foundation for Biomedical Research)was stirred with 1.35 g, 5.5 =moles, p-nitrophenyl nicotinate in 60 mlDMF for 120 hours in room temperature. The mixture was then filtered,evaporated, treated with Et₂ O in ice bath and filtered again.Recrystallization was done by heating in 12 ul EtOH and adding 18 ml hotH₂ O. This produced a clear solution from which crystals separated oncooling. This procedure was repeated twice. Yield: 0.98 g, 50%.Purity>95%. M.p.>220° C. NMR(DMSO d₆): 1.46,s,9H,t-butoxy group;1.9-1.48,m,11H,ring CH₂, ring CH in position 1 and B--CH₂ ; 3.72,m,1H,ring CH in position 4; 3.95,m, 1H, a--CH; 7.48,m,1H,py H⁵ ; 8.16,m,1H,py H⁴ ; 8.67,m, 1H,py H⁶ ; 8.96,m,1H,py H².

BOC-D-cis-NACAla. 5 mmoles BOC-D-cis-3(4aminocyclohexyl)alanine (source:as above) and 5.5 mmoles p-nitrophenyl nicotinate were allowed to reactin DMF as above. Reaction time: 25 hours. Purification was achieved byEt₂ O treatment as above and silica gel chromatography on a 4.5×32 cmcolumn using the solvent system CHCl₃ :MeOH:py:HOAc=75:10:10:5. Yield1.3 g, 61%, amorphous powder. R_(f) (column system)=0.58. NMR (CDCl₃):1.44,s,9H,t-butoxy group; 1.95-1.45,m,11H,ring CH₂, ring CH in position1 and B--CH₂ ; 4.22,m, 1H,a--CH; 4.35,m, 1H,ring CH in position 4: 7.35,8.24, 8.63 and 8.98, 1H each, assignments as previous compound.

BOC-IOrn(Z). This compound was prepared from BOC-Orn(Z) by reductivealkylation with acetone and H₂ /Pd as described by Prasad et al. (23)followed by conversion to the Nd- Z derivative with benzyl chloroformatein aqueous alkali (Schotten-Baumann conditions). Purification wasachieved by chromatography on silica gel with CHCl₃ /MeOH 85:15. R_(f)(CHCl₃ ;MeOH:HOAc=85:15:3)=0.8. NMR(CHCl₃): 1.10,d,6H, isopropyl CH₃ ;1.40,s,9H,t-butoxy group; 1.7-1.5, m,4H,B,y--CH₂ ; 3.09,m,2H, d--CH₂ ;4.2,m, 1H,a--CH; 5.10,s,2H,benzyl CH2; 7.3,m,5H,aromatics.

BOC-CypLys(Z). 2.04 g BOC-Lys(Z) was dissolved in 8 ml of cyclopentanoneand 32 ul H₂ O containing 0.22 g NaOH. Hydrogenation was performed inthe presence of 0.4 g 10% Pd/C at 50-60 psi in a Parr apparatus. After 4hours the hydrogenation was interrupted and 2 ml 0.5 M NaOH and 10 ulMeOH were added. The hydrogenation was then continued for 16 hours at50-60 psi. Then filtration and evaporation. The residue was dissolved in75 ml H₂ O and the aqueous phase extracted with three times with Et₂ Oand once with hexane. The pH was then brought to 6-7 with HCl and thesolution evaporated in rotary evaporator, bath temperature 40° C. Theresulting product was then converted to the Z-derivative using benzylchloroformate in aqueous NaOH (Schotten-Baumann conditions). Yield: 1.3g, 58% overall. R_(f) (n-BuOH:py:HOAc:H₂ O=30:10:3:12) =0.69.Purity>95%. NMR (CDCl.sub. 3): 1.45,s,9H,t-butoxy group;1.95-1.35,m,14H,ring CH₂ +B,y,d--CH₂ ; 3.13,broad t,2H,E--CH₂ ;4.34-4.05,m,2H,a--CH+ring CH; 5.13,s,2H,benzyl CH₂ ; 7.35,m,5H,aromaticprotons.

BOC-Me₂ Lys, D- and L-. These compounds were prepared by hydrogenolysisof the corresponding Z- or Cl-Z-derivatives in the presence of 37%formaldehyde essentially as described by L. Benoiton (22) for the N^(a)-acetyl analog. Purification was achieved by chromatography on silicagel with the solvent system n-BuOH:py:H₂ O=2:2:1. The yields are 40-65%and the products are amorphous. NMR (CDCl₃): 1.41,s,9H,t-butoxy group;1.9-1.5,m,6H,B,y,d--CH₂ ; 2.6,s,6H,N(CH₃)₂ ; 2.8,m,2H,E--CH₂ ; 4.03,m,1H,a--CH.

BOC-D-AnGlu. 0.62 g, 3 mmoles, DCC was added to the ice-cooled solutionof 1.10 g, 3 mmoles, BOC-D-glutamic acid a-benzylester and 0.39 g, 3mmoles, p-anisidine in 25 ml CH₂ Cl₂. The reaction mixture was stirredwhile warming up to room temperature and then another 17 hours. Thedicyclohexylurea was then filtered off and CHCl₃ added to a total volumeof 125 ml. This solution was extracted with 2×1N H₂ SO₄, H₂ O, saturatedNaHCO₃, 2×H₂ O and dried (MgSO₄). Evaporation and recrystallization fromEtOH gave 0.99 g, 74% product, m.p. 129.5°-131° C. Rf (4% MeOH inCHCl₃)=0.53. This product was dissolved in 30 ml MeOH and 10 ul EtOH andhydrogenated in the presence of 0.3 g Pd/C at 50 psi for 2.5 hours.Filtration and evaporation gave a quantitative yield of BOC-D-AnGlu. Notcrystalline. Purity> 98%. NMR (CDCl₃): 1.45,s,9H,t-butoxy group;2.35-1.95,m,2H,B--CH₂ ; 2.6-2.4,m,2H,y--CH₂ ; 3.76,s,3H,OCH₃ ; 4.3,m,1H,a--CH; 6.82 and 7.42, broad d, 2H each, aromatic protons.

BOC-Me₃ Arg. First, N,N,N',S-tetramethylisothiourea was prepared by theprocedure of Lecher and Hardy (19). B.p. (15 mm)=74° C, lit(above) 68°C. at 11 mm. BOC-Orn,9 mmoles, and teramethylisothiourea, 10 mmoles,were dissolved in 15 ml DMF and 2 ul triethylamine and incubated at 100°C. for 2 hours and at room temperature for 10 hours. Then the reactionmixture was evaporated to dryness and passed through a silica gel columneluted by iprOH:triethylamine:H₂ O=42:6:13. The white solid so obtainedwas dissolved in H₂ O and the solution was acidified with 6N HCl andlyophilized to give 5.5 mmoles product. R_(f) (column eluant)=0.50. NMR(D₂ O): 1.42,s,9H,t-butoxy group, 2.80,m, 1H,a--CH; 2.89,s,3H, CH₃ onguanidino group; 2.96,s,6H, (CH₃)₂ N; 3.25,t,2H,d--CH₂ ;1.50,m,4H,B,y--CH₂.

BOC-Dpo. From 10 mmoles arginine hydrochloride and 1.72 g sodiumhydrogen carbonate dissolved in 17 ml H₂ O, 28.6 ul acetylacetone and28.6 ml EtOH was obtained 7.5 mmoles Dpo following the procedure ofF.-S. (20). The product was then converted to the correspondingBOC-derivative using di-t-butyl dicarbonate in 50% aqueous dioxane inthe presence of sodium hydroxide. This reaction proceeds in essentiallyquantitative yield. R_(f) (nBuOH:HOAc:H₂ O=4:1:2)=0.63. NMR (CDCl₃):1.45,s,9H,t-butoxy group; 1.9-1.5,4H,B,y--CH₂ ; 2.33,s,6H,CH₃ ;3.46,m,2H,d--CH_(b) 2 ; 4.24,m, 1H,a--CH; 6.35,s,1H, aromatic H. L- andD- forms react similarly.

BOC-D-Et₂ hArg. This compound was prepared by the method of Nestor andVickery, U.S. Pat. No. 4,530,920, Jul. 23, 1985. R_(f) (nBuOH:HOAc:H₂O=4:1:2)=0.52.

SYNTHESIS OF THE PEPTIDES

The peptides of the present invention were synthesized by the solidphase method using a Beckman Model 990 Peptide Synthesizer. (1, 11) Thebenzhydrylamine hydrochloride resin (BHA-resin) was used as a solidsupport. The program of the synthesizer was divided into subprograms.

1. Deprotection: 1. CH₂ Cl₂ (2×wash, 1 or 2 min); 2. 50% TFA in CH₂ Cl₂containing 0.1% indole (1×wash, 1 or 2 min); 3. 50% TFA in CH₂ Cl₂containing 0.1% indole (deprotection, 20 min); 4. CH₂ Cl₂ (2×wash).

2. Neutralization: 1. CH₂ Cl₂ (2×wash, i or 2 min); 2. DIEA (10% in CH₂Cl₂) (2×wash, 1 or 2 min); 3. DIEA (10% in CH₂ Cl₂) (neutralization, 5min); 4. CH₂ Cl₂ (2×wash, 1 or 2 min).

3. DCC Coupling: 1. CH₂ Cl₂ (2×wash, 1 or 2 min); 2. amino acid solutionin CH₂ Cl₂ (delivery, transfer, mix, 5 min); 3. DCC (10% in CH₂ Cl₂,(delivery and mix, 180 min); 4. CH₂ Cl₂ (2×wash, 1 or 2 min).

4. Active Ester Coupling: not used.

5. Final Wash: 1. CH₂ Cl₂ (2×wash, 1 or 2 min); 2. i-PrOH (3×wash, 1 or2 rain); 3. DMF (3×wash, 1 or 2 rain); 4. CH₂ Cl₂ (3×wash, 1 or 2 rain).

6. Wash after TFA Treatment: 1. CH₂ Cl₂ (2×wash, 1 or 2 min); 2. i-PrOH(2×wash, 1 or 2 min); CH₂ Cl₂ (3×wash, 1 or 2 min).

7. Acetylation: 1. CH₂ Cl₂ (2×wash, 1 or 2 min); 2. 25% Ac₂ O and Py inCH₂ Cl₂ (1×wash, 1 or 2 min); 3. 25% Ac₂ O and Py in CH₂ Cl₂(acetylation, 20 min); 4. CH₂ Cl₂ (2×wash, 1 or 2 min).

The first amino acid was attached to the resin by the program sequence2-3-5. Before placing the resin into the reaction vessel, the resin waswashed in a separatory funnel with 25 ul CH₂ Cl₂ /g resin to remove thefine particles. In all couplings, usually a 3-4 fold excess of theBoc-amino acid over the nitrogen content of the resin was used. Thisprocedure generally resulted in a complete coupling reaction. If apositive ninhydrin color reaction was observed, a second coupling wasperformed (program sequence 3-5). Then, the resin was acetylated(program sequence 7-5).

The next amino acid was attached by the program sequence 1-6-2-3-5. ForDeC coupling, all amino acids were dissolved in CH₂ Cl₂. Acetylation ofthe amino acid residue in position 1 was performed using the programsequence 1-6-2-7-5. The volume of the solvents and the reagents used forthe washing and the performing of the chemical reactions was about 10ul/g resin.

CLEAVAGE OF THE PEPTIDES FROM THE RESIN

After all of the amino acids had been coupled, the peptide resin wasdried overnight, in vacuo. The resin was then treated withdouble-distilled liquid hydrogen fluoride (10 ul/g resin) containing10-25% distilled anisole or p-cresol for 1 hour at 0° C. Then, the HFwas evaporated under reduced pressure and the residue was driedovernight, in vacuo, by an oil pump. The mixture was then extractedseveral times with Et₂ O (25 ul/g resin), then with aqueous. HOAc, 30%,50%, 10%, and once with 25 ul distilled, deionized water. The combinedaqueous solution was lyophilized to yield the crude peptide.

PURIFICATION AND CHARACTERIZATION OF THE PEPTIDES

Most peptides were purified by silica gel chromatography (1×60 cmcolumn) using one of the solvent systems nBuOH:HOAc:H₂ O=4:1:2 or 4:1:5upper phase or nBuOAc:nBuOH:HOAc:H₂ O=2:8:2:3 followed by gel filtrationover Sephadex G 25 with 6% HOAc as the eluant. In the case ofunsatisfactory purity after this procedure the peptides were furtherpurified by semipreparative HPLC using a Waters liquid chromatographequipped with a 660 solvent programmer. A 1.2×25 cm m-Bondapak C₁₈column was used with the solvent system A=0.1 M NH₄ OAc pH 5.0 and B=20%A+80% CH₃ CN. Different gradients of increasing amounts of B in 15-25minutes were employed to effect purification.

An alternate purification scheme has been gel filtration over SephadexG-25 with 6% HOAc followed by chromatography over Sephadex LH 20(2.5×100 cm) with the solvent system H₂ O:nBuOH:HOAc:MeOH=90:10:10:8. Ifnecessary, the latter procedure was repeated 1-2 times.

The purity of the peptides was assessed by thin layer chromatography onMerck silica gel plates in at least four different solvent systems. Thespots were developed with the chlorine/o-tolidine reagent. The equipmentwas the one described above except that an analytical m-Bondapak C₁₈column (3.9 mm×30 cm) was used.

Amino acid analyses were performed on a Beckman model 118 CL amino acidanalyzer. Samples of about 0.5 ug were hydrolyzed in 6N hydrochloricacid in sealed glass tubes for 24 hours at 110° C. The residue was thenevaporated and dissolved in citrate buffer, pH 2.2 and applied to theanalyzer.

BIOASSAYS OF THE PEPTIDES AND DISCUSSION OF DATA

The antiovulatory activity, AOA, in rats was determined as described byHumphries et al. (12). Antiovulation assays were performed by counting,on estrus, the number of ova shed by 4-day cycling rats after a singlesc injection of the LH-RH analogue in corn oil was administered between12 and 12:30 p.m. on proestrus. The control rats received 0.3 mL of thevehicle. The wheal test was performed by intradermally injecting 10 ugof peptide in 100 ul of saline into anaesthesized rats, measuring theideally circular wheal response and calculating the area. The in vitrohistamine release test was done as described by Karten et al. (4). Asuspension of rat mast cells was added to increasing concentrations ofpeptide and the mixture was incubated for a few minutes followed bycentrifugation to collect the histamine in the supernatant. A PIPES(piperazine-N,N'-bis[2-ethanesulfonic acid]) medium buffered at pH 7.4containing NaCl 119 mM, KC1 5 mM, PIPES 25 mM, NaOH 40 mM, glucose 5.6mM, CaCl₂ 1 mM, and 0.1% bovine serum albumin was used. This wasdesignated as PIPES AC. Peptides were dissolved in double distilled H₂ Oat a concentration of 2 mg/ml and stored at -20° C. They were thawedjust prior to testing, diluted in PIPES AC and prewarreed for fiveminutes at 37° C. Peptides studied were stable to heating at 56° C. fortwo hours, and to freezing and thawing.

Peritoneal cells were collected from male Sprague-Dawley rats weighing200 to 250 gm and purchased from Harlan (Madison, Wis.). Aftereuthanasia by CO₂, the peritoneal cavity was washed with 50 ml PIPES ACmedium containing 20 units of heparin. Following centrifugation at 200×gfor eight minutes at 4° C., cells were washed again and finallyresuspended to a concentration of 8 to 24×10⁵ total leukocytes/ml inPIPES AC. This suspension contained approximately 5-10% mast cells.Washed cells were used immediately after collection and were prewarmedfor five minutes at 37° C. prior to pipetting 0.25 ml aliquots into12×75 mm polystyrene tubes containing 0.25 ml of diluted peptide.Mixtures were incubated for 15 minutes at 37° C. and the reactionstopped by centrifugation at 400×g for 15 minutes at 4° C. The cellsupernatants were assayed for histamine content by the automatedfluorometric assay procedure. See, Siraganian, R. P., "An automatedcontinuous flow system for the extraction and fluorometric analysis ofhistamine." Anal. Biochem. 57:383 (1974); and Siraganian, R. P. andHook, W. A., "Histamine release and assay methods for the study of humanallergy," In Manual of Clinical Immunology, 3rd ed., N. R. Rose, H.Friedman, and J. L. Fahey (Eds.), Amer. Soc. for Microbiology,Washington (1986) p. 808.

The results of these bioassays are presented in Table I and other Tablesappended hereto.

Of the 57 peptides in Table I, 21 had an AOA of about 90% or more at adosage of 1 ug in the present assay. 0f the 37 peptides of Table 1tested for histamine release in the rat mast cell assay, 10 had ED₅₀values of 300 or more as compared to 0.17 for the standard compound[N-Ac-D-2-Nal¹, D-4-F-Phe^(2l) , D-Trp³, D-Arg⁶ ]-LHRH. Nine additionalanalogs had ED₅₀ values ranging from 86 to 288, i.e. they do not releasemore histamine than clinically used "superagonists".

Of the thirty-seven peptides of Table 1 tested in the rat mast cellassay, seven (numbers 4, 23, 24, 43 (Antide), 44, 53, 55) had both anAOA of about 90% or more at 1 ug and an ED₅₀ value of about ≧86 ug/ml.This included the potent analog, No. 53, which had 100% AOA at 0.5 ugand 40% AOA at 0.25 ug. The ED₅₀ value for this analog was 93±28. It wasthus demonstrated that high AOA with low histamine release could befound in the analogs of the present invention.

Structural features in common for these seven peptides are: 1) A D-Lysresidue in position 6 which was acylated by the weakly basic nicotinicacid or analogs like picolinic and 6-methylnicotinic acid. 2) Thecorresponding acylated L-Lys residue or the natural Tyr in position 5.3) The alkylated derivatives ILys or IOrn in position 8. 4) Arg isabsent from the sequence.

Two examples of the influence of Arg on histamine release are the pairs43,10 and 4,1. No. 43 (Antide) has the sequence N-Ac-D-2-Na¹ D-pClPhe²sub D-3-Pal³, Ser⁴, NicLys⁵, D-NicLys⁶, Leu⁷, ILs⁸, Pro⁹, D-Ala¹⁰ --NH₂.Its ED₅₀ value is >300. No. 10 is identical in sequence except thatNicLys⁵ is replaced by Arg⁵. This caused the ED₅₀ value to decrease to4.3±0.52. No. 4 has identical sequence as No. 43 except for Tyr inposition 5. Its ED₅₀ value is 133±22. In No 1, ILys⁸ in this sequence isreplaced by Arg⁸ which caused the ED₅₀ value to decrease to 39.2±7. Itthus seems that position 5 is more sensitive than position 8 for Argsubstitution.

In position 8, the alkylated ILys and IOrn residues are superior to Lysand Orn, respectively, both with respect to AOA and histamine release(pairs 3,4 and 6,7). Whether ILYs⁸ or IOrn⁸ is best seems to be sequencedependent.

BIOLOGICAL ACTIVITIES

For the determination of duration of action, the antagonist wasadministered s.c. or orally to 26 days old female rats at a specifictime before administration of the agonist, [D-Qal⁶ ]-LHRH. The serumlevels of rat luteinizing hormone (LH) and rat follicle stimulatinghormone (FSH) were then measured 2 hours after the agonistadministration by RIA. The oral administration was done throughforce-feeding with feeding tubes.

AOA AND HISTAMINE RELEASE DATA

Table IV shows data on AOA and histamine release for analogs containingacylated aminocyclohexylalanine residues. For the analogs with NicLys⁵,D-NACAla⁶, IV-1 and IV-2, (NACAla represents3(4-nicotinoylaminocyclohexyl)alanine), analog 2 with cis-D-NACAla⁶ issomewhat more active, 100% vs. 70% AOA at lug. Analogs IV-7 and IV-8with NicLys⁵, D-PzACAla⁶ (PzACAla represents3(4-pyrazinylcarbonylaminocyclohexyl)alanine) show the opposite order ofactivity. The trans residue has the higher AOA, 88% vs. 25% at lug.

Analogs IV-3 and IV-4 with PicLys⁵, trans and cis PACAla⁶ (PACAlarepresents 3(4-picolinoylaminocyclohexyl)alanine) are equipotent, 50 and54% AOA at 0.5 ug, respectively, whereas in the case of PicLys⁵, transand cis PzACAla⁶ the cis compound is more than twice as active. Theformer, analog IV-5 is about as potent as analogs IV-3 and IV-4 (44% at0.5 ug) while the latter, analog 6, has 100%, 73%, and 29% AOA at 0.5,0.25, and 0.125 ug, respectively. The high potency analog IV-6 is uniquein comparison with the low activity of the structurally similar analogIV-8.

Analog IV-9 has cis-PzACAla⁵, D-PicLys⁶ and although residues 5 and 6are reversed, retained the high potency of analog IV-6, 90% and 67% at0.5 and 0.25 ug, respectively. 5 As for histamine release, all analogstested, in vitro, have lower ED₅₀ values than the parent compounds. TheED₅₀ values range from about 30 to about 60 compared to >300 and 93±11for Antide and analog V-10. The tests for wheal response show a rangefrom 99.5 to 129.6, which is similar to Antide (132.7) and analog V-10(123.0). The lack of correlation between the two tests may primarilyreflect assay variation.

In summary, for the analogs with NicLys⁵, 5 incorporation ofaminocyclohexylalanine derivatives in position 6 resulted in substantialincrease in, in vitro, histamine release and unchanged or lowered AOA.For the PicLys⁵ analogs with the same substitutions there was loweringof AOA potency in all cases except one, where a considerable increasewas observed. The combination PicLys⁵ and cis-D-PzACAla⁶ evidentlypossesses some beneficial structure. Histamine release for the PicLys⁵analogs was increased by 50-100%.

In Table V, are the results from substitutions in position 7 of analogV-10. This position allows some structural freedom although none of thepeptides show higher AOA than analog V-10. Analogs V-12, V-14, and V-16having Aile⁷ (alloisoleucine), Val⁷ and Abu⁷ (2-aminobutyric acid), areequipotent with analog V-10. Analog V-16 with the straight chain Abu⁷ isslightly more potent than analogs V-13 and V-15 with Nle⁷ (norleucine)and Nval⁷ (norvaline), respectively, which should more closely resemblethe natural Leu⁷.

For compound V-17 with the small Ala⁷, the AOA decreased to 60% at 0.5ug. Incorporation of Trp⁷ which is the natural residue in chicken II,salmon and lamprey LHRH's (13-15), gave analog 18 with only 10% AOA at0.5 ug. Trp⁷ may be too large considering the size of the adjacentD-PicLys⁶ and Ilys⁸.

The most interesting feature of Table V is the, in vitro, histaminerelease data. The three analogs with similar AOA potency as analog V-10show markedly diminished histamine release. The ED₅₀ values for analogsV-12, V-14, and V-16 are >300, 213±30 and 273±27, respectively; i.e., a2-3 fold decrease in histamine release is achieved by small changes inside chain structure. Also, the wheal response is diminished for allanalogs compared to V-10.

It was noted earlier (1) that whether ILys or IOrn is the bestsubstituent in position 8 is sequence dependant. To further investigatethis aspect, the IOrn⁸ analogs corresponding to some of the bestpeptides were synthesized and tested. The results in Table VI indicatethat Ilys⁸ may be better. For two of the pairs, analogs VI-10, VI-19 andVI-12, VI-21, ILys⁸ and IOrn⁸ were about equivalent. For the other threepairs, the analogs with ILys⁸ were more active, but the differences werenot large. The largest difference was for the pair with Val⁷, where theILys⁸ -analog VI-14 showed 90% AOA at 0.5 ug vs. 57% for the IOrn⁸-analog VI-20.

Analog VI-19 was tested, in vitro, for histamine release. The ED₅₀ valueis 42±3.1; i.e., the histamine release is 2-fold that of the analog withone more CH₂ unit. The wheal response did not change conspicuouslyexcept for the Aile⁷ and IOrn⁸ analog 21 which had the low value of78.6±4.5 compared to the ILys analog 12 which had 97.9±2.9.

DURATION OF ACTION

Table VII shows the duration of action of Antide and two analogs. WhenAntide was injected 44 hours before 50 ng of [D-Qal⁶ ]-LHRH (Qalrepresents 3(3-quinolyl)alanine), a superagonist, at doses of 3, 10, and30 ug, significant reductions in serum LH were observed at the twohigher doses. The LH decreased from 113±11 to 46±12 and 5±0.7 ng/ul.Serum FSH was also decreased, most significantly from about 300 to about3000 ng/ml at 30ug.

Analog VII-24, [Tyr⁵ ]-Antide, and analog IV-6 were similarly injected24 hours before the agonist. Analog VII-24 showed high activity,reducing the LH level to 19±4, 3±0.4 and 0.3±0.03 ng/ul at doses of 3,10, and 30 ug, respectively. The corresponding figures for analog IV-6are 42±7, 15±3, and 3.4±2 ng/ul. This is interesting since in theantiovulatory assay analog IV-6 is considerably more potent, 73% at 0.25ug vs. 45% at 0.5 ug. Perhaps, analog IV-6 is enzymatically degradedfaster than analog VII-24. The long duration of action of these analogss.c. may also be due to "depot" effects at the site of injection.

ORAL ACTIVITY

Table VIII shows the duration of action of Antide after oraladministration. Forty-eight hours after administration of 100 or 300ugdose levels of Antide, there were significantly reduced levels of LHwhich had been released by 5 ng of [D-Qal⁶ ]-LHRH s.c. Reductions from21±3 to 4±0.8 and 8±2 ng/ul, respectively, were observed. The resultsare about the same in the -24 hour experiment (9±2 and 6±0.3 ng/ul).Antide appears to possess considerable resistance towards degradingenzymes. When Antide was given 2 hours before the agonist, a strongdecrease in LH levels was observed. At a dose of 30 ug, a significantlowering of the LH level to 6±1 ng/ul was seen. At 100 and 300 ug, thelevels were 1±0.3 and 0.4±0.4 ng/ul, i.e., very low levels. When 10 ngof agonist was used, the results are qualitatively very similar.

For comparison, the last three entries in Table VIII are fromexperiments with [N-Ac-D-pClPhe¹,2, D-Arg⁶, D-Ala¹⁰ ]-LHRH, VIII-25 ananalog that has been reported to have oral activity, (16). These datashow that Antide is more active than VIII-25, since a dose of 30 uggiven 2 hours before the agonist reduced the LH level from 44±4 to 22±4ng/ul (p<0.01}. The value for analog VIII-25 is 39±6 (NS). At 100 ug,the corresponding numbers are 7±3 (p<0.001) and 26±7 (p<0.05). The FSHlevels were, in general, lowered when Antide was administered at -2hours at 100 or 300 ug dose levels.

The results in Table IX show that there is no significant differencebetween administration of Antide in water or in corn oil.

Antide has also been tested orally in the antiovulatory assay (Table X).The AOA values at 300, 600, and 1200 ug dose levels are 18, 73, and 100%respectively. Expressed as rats ovulated/total rats, the numbers are9/11, 3/11, and 0/11. For analog VIII-25, the numbers 9/11, 4/11, and0/11 have been reported at dose levels of 500, 1000, and 2000 ug,respectively, (16). Antide was about twice as active as analog VIII-25.

Table XI shows a comparison of the oral activities of Antide and fouranalogs. One was as active as Antide, one was considerably less activeand two were less active at low doses (30 and 100 ug) and about asactive at 300 ug.

After a 15 ng s.c. dose of [D-Qal⁶ ]-LHRH, the LH level rose to 91±4.6ng/ul. At oral dose levels of 30, 100, and 300ug of Antide, reducedlevels of LH of 75±3, 20±4, and 5±1 ng/ul, respectively, were recorded.Analog 4 with PicLys⁵, and D-PACAla⁶ showed no significant reduction ofLH at 30 and 100ug levels, but there was a reduction to 51±6 ng/ul at a300 ug dose.

Analog V-12 with PicLys⁵, D-PicLys⁶, and Aile⁷ and analog IV-6 withPicLys⁵, cis-D-PzACAla⁶ are less active than Antide at 30 and 100 ug,but were equally active at 300 ug. Both of these peptides weresubstantially more active than Antide in the s.c. antiovulatory assay.

Analog 26 was equipotent with Antide. This is not surprising since theonly structural difference between these analogs is a pyrazine insteadof a pyridine moiety in the N^(E) -acyl group of the D-Lys⁶ residue.

Table XI and XII also shows results with Antide, for example, when 50 ngof the agonist was used. Comparison of these results with the data fromthe experiments using 15 ng of agonist shows a dose-responserelationship which is expected from competitive antagonism. Using 15 ngof agonist, 100 and 300ug of Antide reduced the LH level from 115±15ng/ul to 20±4 and 5±1 ng/ul respectively, while in the experiments using50 ng of agonist, 300 and 900 ug of Antide reduced the LH to the samelevel (19±3 and 5.3±1.2 ng/ul).

Table XIII shows the biological effects of Antide in a dispersedpituitary cell culture system.

The structures and biological activities of certain preferred LHRHanalogs inhibiting more than 50% of ovulatory activity at a dose of 0.25ug are shown in Table XIV.

It is proposed that Antide and other antagonists of the presentinvention may be utilized to induce a state of reversible medicalcastration that will be of value in the treatment of a rather largenumber of diseased states such as endometriosis, uterine fibroids andhormonal dependent cancers (prostate, breast). In some patientstemporary inhibition of the function of the gonads with Antide, forexample, while the patient is receiving chemotherapeutic agents and/orirradiation may prevent or minimize adverse effects of these agents onthe gonads and thus help to preserve future fertility. Therapeuticexamples would be irradiation during bone marrow transplantation,cervical carcinoma, metastatic thyroid and uterine carcinoma, possiblythyrotoxicosis, etc. during chemotherapy for disseminated lupuserythematosus and certain stages of organ transplantation. Morephysiological usages of the antagonists of the present invention such asAntide would be to inhibit fertility in both females and males.

More unique possible usages of Antide or other decapeptides of thepresent invention would be to modify sexual behavior during selectdisease states. Such disease states could involve patients with AIDS,the aggressive behavior of sex offenders in prisons or aggressiveadolescents confined to corrective institutions. It is also possiblethat high serum gonadotrophin levels of post-menopausal women may inducefunctional abnormalities in fat cells that cause weight gain or in bonecells that play a role in accelerated osteoporosis. These functionalabnormalities could potentially be reduced with administration of Antideby inhibiting the high LH and/or FSH level in serum of post menopausalwomen.

Selective LH-RH antagonists mainly with charged amino acid substitutionsin position 6 and/or 8 of the decapeptides probably stimulate histaminerelease by a direct effect on mast cells to release histamine whileother LH-RH antagonists like Antide do not. It is thus proposed that themast cell-stimulating antagonists applied locally to wounds of the skinmay accelerate healing while non-histamine stimulating antagonists mayprevent some of the allergic reactions which occur in humans.

To delay the onset of puberty in short stature children byadministration of Antide with and without concomitant administration ofGH or GH-releasing peptides is proposed as a unique method to enhancebody height. The presence Of gonadal hormones fuse the epiphysis of longbone and prevent their further elongation. This approach should extendand augment the use and effectiveness of GH and GH-releasing peptides.

The administration of LH-RH antagonists of the present invention acutelyinhibits the function of the gonads within 24 hours. Continuousadministration of LH-RH superagonists also inhibits the function of thegonads but this is only after several days of stimulating the gonads tohyperfunction. Such superagonist administration introduces a number ofpotential undesirable clinical problems in patients with prostatecancer, endometriosis, uterine fibroids as well as with sex offendersand those subjected to a temporary induction of medical castration. Forthese reasons it is proposed that LH-RH antagonists will be moredesirable agents than LH-RH agonists for introducing a reversible stateof medical castration. At the diagnostic level, such as differentiatingthe anatomic source of steroid secretion from the adrenal versus theovary or to reveal the degree of calcium excretion dependency-on gonadalsteroid hormones, the rapid onset of inhibiting gonadal function withLH-RH antagonists makes them an unequivocally superior agent over LH-RHagonists. It is proposed that, in every clinical situation where LH-RHsuperagonists have been utilized to inhibit gonadal function, the LH-RHantagonists will be the agents of choice.

The references in the following list are incorporated by referenceherein.

                                      TABLE I                                     __________________________________________________________________________    ANTAGONISTS OF LHRH BASED UPON                                                [( ).sup.1,D-pClPhe.sup.2,( ).sup.3,Ser.sup.4,( ).sup.5,(                     ).sup.6,Leu.sup.7,( )8,Pro.sup.9,D--Ala.sup.10 ]--NH.sub.2                           Compound                             AOA %/μg                                                                           Wheal                                                                                ED.sub.50          NO.                                                                              IBR #                                                                             ( ).sup.1 ( ).sup.3                                                                           ( ).sup.5                                                                           ( ).sup.6 ( ).sup.8                                                                          0.5                                                                             1.0                                                                              2.0                                                                              mm.sup.2 /10                                                                         μg/ml           __________________________________________________________________________    ANALOGS WITH D-NICLYS IN POSITION 6                                           1. 22396                                                                             N--Ac--D-2-Nal                                                                          D-3-Pal                                                                             Tyr   D--NicLys Arg  60                                                                              100                                                                              -- 85     39.2 ±                                                                     7                  2. 24753                                                                             "         "     "     "         Me.sub.3 Arg                                                                       --                                                                              -- --        39.9 ±                                                                     7                  3. 24825                                                                             "         "     "     "         Lys  --                                                                              27 -- 119.5 ±  3.2           4. 24315                                                                             "         "     "     "         ILys 45                                                                              89 100                                                                               79.0                                                                                133 ±                                                                      22                 5. 24443                                                                             "         "     "     "         Me.sub.2 Lys                                                                       --                                                                              90 100                                                                              122.7  18.4               6. 24748                                                                             "         "     "     "         Orn  --                                                                              -- 67 129.4                                                                                19.3 3.3           7. 24756                                                                             "         "     "     "         IOrn 22                                                                              71 --  92.2                                                                                >300 2.9           8. 24199                                                                             "         "     Arg   "         Arg   0                                                                              42 -- 146.8                     9. 24446                                                                             "         D--Tyr                                                                              "     "         "    33                                                                              -- -- 113.2                                                                                1.73 5.6           10.                                                                              25335                                                                             "         D-3-Pal                                                                             "     "         ILys 43                                                                              17 -- 196.9                                                                                4.3 ±                                                                      0.52                  24931                                                                             "         "     Me.sub.3 Arg                                                                        "         "    --                                                                              -- 44 140 ± 7.0                 25506                                                                             "         "     Dpo   "         "    56                                                                              -- -- 110 ± 3                   24543                                                                             "         "     ILys  "         "    --                                                                              89 -- 132.7                                                                                20.3 0                24545                                                                             "         "     His   "         Arg  --                                                                              89 -- 139.7 ± 0                 24593                                                                             "         "     3-Pal "         "    --                                                                              100                                                                              -- 146.4 ± 3.6               25383                                                                             "         "     "     "         ILys --                                                                              75 -- 132.8                                                                                86 ±0                                                                      28*                   25384                                                                             "         "     "     "         IOrn --                                                                              100                                                                              -- 139.9                                                                                55 ±2                                                                      13*                   25144                                                                             "         "     Ile   "         ILys --                                                                              82 -- 147.7                                                                                324 ±                                                                      20                    25145                                                                             "         "     "     "         IOrn --                                                                              55 -- 116.5                                                                                151 ±                                                                      75                 20.                                                                              25333                                                                             "         "     NicOrn                                                                              "         "    --                                                                              73 -- 113.6                                                                                57 ±.9                                                                     13                    25509                                                                             "         "     DMGLys                                                                              "         ILys 20                                                                              -- -- 110                                                                                  34 ±                                                                       1.1                   25510                                                                             "         "     PicLys                                                                              "         "    64                                                                              100                                                                              -- 116                                                                                  39 ±3                                                                      1.0                   25337                                                                             N--Ac--D-pClPhe                                                                         "     Tyr   "         "    --                                                                              100                                                                              -- 139.9                                                                                198 ±                                                                      33*                   25338                                                                             N--Ac--D--Cl.sub.2 Phe                                                                  "     "     "         "     0                                                                              89 -- 103.9                                                                                311 ±                                                                      65*                ANALOGS WITH NICLYS IN POSITION 5                                                22495                                                                             N--Ac--D-2-Nal                                                                          D-3-Pal                                                                             NicLys                                                                              D-3-Pal   Arg   0                                                                              -- -- 112                          24544                                                                             "         "     "     D--His    "    --                                                                              100                                                                              -- 146.7 ± 3.6               24754                                                                             "         "     "     D--ILys   "    --                                                                              56 73 196.9 ± 4.1               25334                                                                             "         "     "     D--Dpo    ILys 40                                                                              100                                                                              -- 165.2                                                                                6.7 ±                                                                      2.2                   25332                                                                             "         "     "     D--BzLys  "    --                                                                              50 -- 119.6                                                                                >300 6.7           30.                                                                              25507                                                                             "         "     "     D--Et.sub.2 hArg                                                                        "    --                                                                              67 -- 123 ± 5.8                 25589                                                                             "         "     "     D--PicLys "    36                                                                              -- -- 120                                                                                  60 ±                                                                       1.4                   25588                                                                             "         "     "     D--AnGlu  "    --                                                                              67 -- 113                                                                                  >300 7                25647                                                                             "         "     "     trans-D--NACAla                                                                         "    --                                                                              70 -- 119.5 ± 3.2               25648                                                                             "         "     "     cis-D--NACAla                                                                           "    --                                                                              100                                                                              -- 113.6                                                                                37 ±.9                                                                     1.1                   25591                                                                             "         "     "     D--Me.sub.2 Lys                                                                         "    --                                                                              82 -- 111                                                                                  262 ±                                                                      23                    25649                                                                             "         "     "     D-PzcLys  "    78                                                                              92 -- 122.2 ± 5.1            ANALOGS WITH NICLYS IN POSITION 8                                                24749                                                                             N--Ac--D-2-Nal                                                                          D-3-Pal                                                                             Tyr   D-Arg     NicLys                                                                             --                                                                              -- 88 136.3                                                                                14.2 6.8              24771                                                                             "         "     Arg   D-3-Pal   "     0                                                                              -- --  99.0 ± 10.3              24824                                                                             "         "     Tyr   D--ILys   "    --                                                                              -- 100                                                                              122.8 ± 5.8            ANALOGS WITH NICLYS AND D--NICLYS                                             IN POSITIONS 5, 6 OR IN POSITION 8, 6 OR IN POSITONS 3, 6                     40.                                                                              24594                                                                             N--Ac--D-2-Nal                                                                          D-3-Pal                                                                             NicLys                                                                              D--NicLys Arg  22                                                                              100                                                                              -- 126.2 ± 8.8               24987                                                                             "         "     "     "         Me.sub.3 Arg                                                                       --                                                                              -- 100                                                                              150.9 ± 14.0              25143                                                                             "         "     "     "         Dpo  --                                                                              18 -- 113.6                                                                                <300 11.1             24542                                                                             "         "     "     "         ILys 36                                                                              100                                                                              100                                                                              132.7                                                                                300. 0                24933                                                                             "         "     "     "         lOrn 88                                                                              100                                                                              -- 136.0                                                                                206 ±                                                                      64                    25078                                                                             "         "     "     "         CypLys                                                                             --                                                                              64 -- 147.0                                                                                171 ±                                                                      49                    24540                                                                             "         "     Tyr   "         NicLys                                                                             --                                                                               0 --  82.6                                                                                300. 2.8              24745                                                                             "         "     His   "         "    --                                                                              -- 18 136.3 ± 6.8               24746                                                                             "         "     ILys  "         "    --                                                                              30 -- 132.8 ± 5.9               24597                                                                             "         D--NicLys                                                                           Tyr   "         Arg  --                                                                              89 -- 101.0 ± 6.0                                                                  Wheal                                                                         Area                           Compounds                        AOA %/μg   mm.sup.2 /                                                                         ED.sub.50          NO.                                                                              IBR #                                                                             ( ).sup.1 ( ).sup.3                                                                          ( ).sup.5                                                                           ( ).sup.6                                                                            ( ).sup.8                                                                          0.25                                                                             0.5                                                                              1.0                                                                              2.0                                                                             10.0                                                                             10                                                                                 μg/ml           __________________________________________________________________________    MISCELLANEOUS ANALOGS                                                         50.                                                                              24596                                                                             N--Ac--D-2-Nal                                                                          D-3-Pal,                                                                           NicLys,                                                                             D--NicLys,                                                                           NicLys                                                                             -- --  0 --                                                                              -- 122.8 ±                                                                    5.7                        24934                                                                             "         "    "     NicLys Ilys -- -- -- --                                                                               8 123                                                                                >300                                                                     5.9                        25146                                                                             "         "    INicLys                                                                             D--INicLys                                                                           "    -- 63 91 --                                                                              -- 140.3                                                                              15 ±                                                                  13.9 8.2                   25147                                                                             "         "    PicLys                                                                              D--PicLys                                                                            "    40 100                                                                              90 --                                                                              -- 123.0                                                                              93 ±                                                                  0    28                    25385                                                                             "         "    Arg   D--BzLys                                                                             "    -- -- 63 --                                                                              -- 169.0                                                                              8.7 ±                                                                 7.7  3*                    25386                                                                             "         "    MNicLys                                                                             D--MNicLys                                                                           "    -- 56 100                                                                              --                                                                              -- 126.1                                                                              >300*                                                                    6.7                        25508                                                                             "         "    DMGLys                                                                              D--BzLys                                                                             "    -- -- 100                                                                              --                                                                              -- 136.6.7                                                                            24 ±                                                                       0.3                   25650                                                                             "         "    PzcLys                                                                              D--PzcLys                                                                            "    -- 17 -- --                                                                              -- 110.2                                                                              288 ±                                                                 8.1  30                 __________________________________________________________________________     *In this test series, the standard compound had an E.sub.D 50 value of        0.46 instead of the usual 0.1-0.2.                                       

    TABLE II      IBR # Sequence       ANALOGS WITH PicLys.sup.5, D-PicLys.sup.6 58 26100 N--Ac--D-Cl.sub.2     Phe, D-pClPhe, D-3-Pal, Ser, PicLys, D-PicLys, Leu, ILys, Pro, D--Ala--NH     .sub.2 59. 25807 N--Ac--D-2-Nal D--Cl.sub.2 Phe " " " " " " " " 60.     26364 " D-3-dal D-pClPhe " " " " IOrn " " 61. 26119 " D-pClPhe D-3-PzAla     " " " " " " " 62. 26177 " " D--Trp " " " " ILys " " 63. 25934 " "     D-3-Pal " " " Val " " " 64. 26118 " " " " " " " IOrn " " 65. 25936 " " "     " " " Aile, ILys " " 66. 26178 " " " " " " " IOrn " " 67. 25990 " " " "     " " Abu ILys " " 68. 26179 " " " " " " " IOrn " " 69. 25935 " " " " " "     Trp ILys " " 70. 25988 " " " " " " Nle " " " 71. 25989 " " " " " " Nval     " " " 72. 26020 " " " " " " Ile " " " 73. 26099 " " " " " " Ala " " "     74. 26346 " " " " " " Abu Arg " " 75. 25937 " " " " " " Leu ILys Pip "     76. 26019 " " " " " " " " Pro, D--Abu--NH.sub.2 77. 25933 " " " " " " "     Iorn " D--Ala--NH.sub.2 Analogs with PicLys.sup.5. 78. 26349 N--Ac--D-2-N     al D-pClPhe D--TinGly Ser PicLys c-D--PzACAla Leu ILys Pro D--Ala--NH.sub     .2 79. 26324 " " D-3-PxAla " " " " " " " 80. 25897 " " D-3-Pal " " " " "     " " 81. 26181 " " " " " " " IOrn " " 82. 26325 " " " " " " Val ILys " "     83. 26366 " " " " " " Phe " " " 84. 26347 " " " " "  " Leu Arg " " 85.     26348 " " " " " " " ILys " D--Ser--NH.sub.2 86. 26383 " " " " " " " " "     NHEt 87. 26323 " " " " " c-D-PmACAla " " " D--Ala--NH.sub.2 Analogs with     D-PicLys.sup.6 88. 26180 N--Ac--D-2-Nal D-pClPhe D-3-Pal Ser c-PzACAla     D--PicLys Leu ILys Pro D--Ala--NH.sub.2 89. 26381 " " " " HOBLys " Abu "     " " 90. 26382 " " " " Cit " " " " " 91. 26363 " " " " Tyr " Leu IOrn " " A     nalogs with NicLys.sup.5. 92. 25805 N--Ac--D-2-Nal D-pClPhe D-3-Pal Ser     NicLys t-D--PzACAla Leu ILys Pro D--Ala--NH.sub.2 93. 25806 " " " " "     c-D--PzACAla " " " " 94. 26345 " " " " " D--NicLys NMeLeu " " " 95.     25991 " " " " " D--PzcLys Leu IOrn " " Miscellaneous Substitutions in     Positions 5 and 6. 96. 25808 N--Ac--D-2-Nal D-pClPhe D-3-Pal Ser MPicLys D     --M--PicLys Leu ILys Pro D--Ala--NH.sub.2 97. 26322 " " " " PmcLys     D--PmcLys " " " " 98. 26326 " " " " c-PzACAla c-D--PzACAla " " " " 99.     26417 " " " " " D-3-PzAla " Arg " " 100. 26418 " " " " Tyr c-D--PzACAla     " ILys " " 101. 26365 " " " " " " " " " D--Ser--NH.sub.2 Analogs Being     Synthesized at This Time. 102.  N--Ac--D-2-Nal D-pClPhe D-3-Pal Ser Arg     D-3-Pal Leu Arg Pro Sar--NH.sub.2 103.  " " " " PicLys c-D--PzACAla "     ILys " " 104.  D-pGlu " D--Phe " Arg D-3-Pal " Arg " D-Ala-NH.sub.2 105.      N--Ac--D-2-Nal " D-3-Pal " c-PzACAla D-PicLys Val ILys "

                  TABLE III                                                       ______________________________________                                        Biological Data.                                                              Analogs with PicLys.sup.5,D--PicLys.sup.6                                                                  In Vitro Hist-                                                                amine Release                                           AOA/μg  Wheal Area ED.sub.50 μg/ml ±                          NO.  IBR #   0.25   0.5  1.0  mm.sup.2 /10 μg                                                                     SEM                                    ______________________________________                                        58.  26100   --     38   --   116.2 ± 3.7                                  59.  25807   --     64   90   139.8 ± 7.1                                  60.  26364   12     --   --   116.2 ± 5.5                                  61.  26119   --     75   --   103.9 ± 3.4                                  62.  26177   --     20   --    71.0 ± 4.3                                  63.  25934   43     90   100   97.9 ± 2.9                                                                         213 ± 30                            64.  26118   --     57   --   119.6 ± 6.6                                  65.  25936   43     89   --    97.9 ± 2.9                                                                         >300                                   66.  26178   --     82   --    78.6 ± 4.5                                  67.  25990   36     100  --    91.0 ± 5.4                                                                         273 ± 27                            68.  26179   --     80   --   101.5 ± 9.3                                  69.  25935   --     10   --    78.5 ± 0                                    70.  25988   20     77   --   107.0 ± 6.0                                  71.  25989   10     100  --    95.3 ± 6.0                                  72.  26020    0     --   --   110.7 ± 2.3                                  73.  26099   --     60   --   103.9 ± 3.7                                  74.  26346   50     88   --   113.2 ± 5.4                                  75.  25937   --      0   100   95.0 ± 0                                    76.  26019   --     78   --   109.9 ± 3.0                                  77.  25933   50     90   100  113.0 ± 0                                    Analogs With PicLys.sup.5                                                     78.  26349    0     --   --    84.6 ± 3.9                                  79.  26324   22     100  --   127.8 ± 4.9                                  80.  25897   73     100  --   122.8 ± 5.7                                                                           28 ± 7.5                          81.  26181   50     100  --   101.6 ± 2.2                                  82.  26325   73     100  --   127.8 ± 4.9                                  83.  26366    0     --   --   116.2 ± 3.2                                  84.  26347   14     --   --   119.6 ±  8.5                                 85.  26348   22     --   --   122.8 ± 5.7                                  86.  26383   25     --   --   119.6 ± 6.6                                  87.  26323   --      9   --   120.4 ± 4.7                                  Analogs With D--PicLys.sup.6                                                  88.  26180   67     90   --    99.5 ± 4.5                                  89.  26381   11     --   --    95.1 ± 5.0                                  90.  26382   11     --   --    89.5 ± 5.5                                  91.  26363    0     --   --   113.2 ± 5.5                                  Analogs With NicLys.sup.5                                                     92.  25805   --     67   88   129.6 ± 8.8                                  93.  25806   --     --   25   101.7 ± 5.0                                  94.  26345   10     --   --   110.5 ± 11.4                                 95.  25991   --     44   --   104.3 ± 10.5                                 Analogs With Miscellaneous Substituents in Positions 5 and 6.                 96.  25808   --     67   91   106.2 ± 4.3                                  97.  26322    0     --   --   130.2 ± 2.5                                  98.  26326   57     100  --   115.5 ± 2.4                                  99.  26417   22     --   --   133.2 ± 11.8                                 100. 26418   22     --   --    95.0 ± 0                                    101. 26365    0     --   --   129.4 ± 3.3                                  ______________________________________                                    

                                      TABLE IV                                    __________________________________________________________________________    Biological Data for [N--Ac--D-2-Nal.sup.1,D-pClPhe.sup.2,D-3-Pal.sup.3,X.s    up.5,Y.sup.6,ILys.sup.8,D--Ala.sup.10 ]-LHRH Analogs                                                         In Vitro Hist-                                                    AOA %/μg amine Release ED.sub.50                                                                 Wheal Area                           NO.                                                                              X      Y        0.125                                                                            0.25                                                                             0.5                                                                              1.0                                                                              μg/ml ± SEM                                                                       mm.sup.2 /10 μg                   __________________________________________________________________________    IV-1.                                                                            NicLys trans-D-NACAla                                                                         -- -- -- 70           119.5 ± 3.2                       IV-2.                                                                            "      cis-D-NACAla                                                                           -- -- 50 100                                                                              37 ± 1.1                                                                             101.8 ± 4.3                       IV-3.                                                                            PicLys trans-D-PACAla                                                                         -- -- 50 -- 64 ± 5.4                                                                             101.0 ± 3.0                       IV-4.                                                                            "      cis-D-PACAla                                                                           -- -- 54 -- 41 ± 5.4                                                                             123.0 ± 5.0                       IV-5.                                                                            "      trans-D-PzACAla                                                                        -- -- 44 -- 39 ± 4.4                                                                             106.3 ± 4.3                       IV-6.                                                                            "      cis-D-PzACAla                                                                          29 73 100                                                                              -- 28 ± 7.5                                                                             122.8 ± 5.7                       IV-7.                                                                            NicLys trans-D-PzACAla                                                                        -- -- 67 88           129.6 ± 8.8                       IV-8.                                                                            "      cis-D-PzACAla                                                                          -- -- -- 25           101.7 ± 5.0                       IV-9.                                                                            cis-PzACAla                                                                          D-PicLys -- 67 90 --            99.5 ± 4.5                       __________________________________________________________________________

                  TABLE V                                                         ______________________________________                                        Biological Data for                                                           [N--Ac--D-2-Nal.sup.1,D-pClPhe.sup.2,D-3-Pal.sup.3,PicLys.sup.5,D--PicLys.    sup.6,X.sup.7,                                                                ILys.sup.8,D--Ala.sup.10 ]-LRRH Analogs.                                                          In Vitro                                                                      Histamine                                                                     Release                                                            AOA %/μg                                                                              ED.sub.50 μg/                                                                        Wheal Area                                      NO.    X       0.25   0.5  1.0  ml ± SEM                                                                           mm.sup.2 /10 μg                    ______________________________________                                         V-10.*                                                                              Leu     40     100   90   93 ± 11                                                                           123 ± 0                            V-11.  Ile      0     --   --           110.7 ± 2.3                        V-12.  Aile    43     89   --   >300    97.9 ± 2.9                         V-13.  Nle     20     77   --           107.0 ± 6.0                        V-14.  Val     43     90   100  213 ± 30                                                                           97.9 ± 2.9                         V-15.  NVal    10     100  --           95.3 ± 6.0                         V-16.  Abu     36     100  --   273 ± 27                                                                           91.0 ± 5.4                         V-17.  Ala     --     60   --           103.9 ± 3.7                        V-18.  Trp     --     10   --           78.5 ± 0                           ______________________________________                                         *From Reference 1                                                        

                                      TABLE VI                                    __________________________________________________________________________    Biological Data for                                                           [N--Ac--D-2-Nal.sup.1,D-pClPhe.sup.2,D-3-Pal.sup.3,PicLys.sup.5,X.sup.6,Y.    sup.7,Z.sup.8,D--Ala.sup.10 ]-LHRH Analogs                                                                In Vitro Hist-                                                       AOA %/μg                                                                            amine Release ED.sub.50                                                                 Wheal Area                              NO.  X       Y  Z  0.25                                                                             0.5                                                                              1.0                                                                              μg/ml ± SEM                                                                       mm.sup.2 /10 μg                      __________________________________________________________________________     VI-10.*                                                                           D-PicLys                                                                              Leu                                                                              ILys                                                                             40 100                                                                               90                                                                               93 ± 11                                                                               123 ± 0                            VI-19.                                                                             "       "  IOrn                                                                             50 90 100                                                                                42 ± 3.1                                                                           113.0 ± 0                            VI-14.                                                                             "       Val                                                                              Ilys                                                                             43 90 100                                                                              213 ± 30                                                                              97.9 ± 2.9                          VI-20.                                                                             "       "  IOrn                                                                             -- 57 --           119.6 ± 6.6                          VI-12.                                                                             "       Aile                                                                             ILys                                                                             43 89 -- >300       97.9 ± 2.9                          VI-21                                                                              "       "  IOrn                                                                             -- 82 --            78.6 ± 4.5                          VI-16.                                                                             "       Abu                                                                              ILys                                                                             36 100                                                                              -- 273 ± 27                                                                              91.0 ± 5.4                          VI-22.                                                                             "       "  IOrn                                                                             -- 80 --           101.5 ± 9.3                          VI-6.                                                                              cis-D-PzACAla                                                                         Leu                                                                              ILys                                                                             73 100                                                                              --   28 ± 7.5                                                                           122.8 ± 5.7                          VI-23.                                                                             "       "  IOrn                                                                             50 100                                                                              --           101.6 ± 2.2                          __________________________________________________________________________     *From Reference 1                                                        

                                      TABLE VII                                   __________________________________________________________________________    Duration of Action of Antide and Two Analogs Subcutaneously*                  Administered.                                                                               0 Time                                                                        ng sc +2 hrs                                                          Injection                                                                          Dose                                                                             [D-3-Qal.sup.6 ]-                                                                   LH ng/ml ±                                                                            FSH ng/ml ±                                 Analog                                                                              Time μg                                                                            LHRH  SEM    p value                                                                           SEM     p value                                __________________________________________________________________________    --    --   -- --     0.4 ± 0.03                                                                       <.001                                                                             143 ± 10                                                                           <.001                                  --    --   -- 50    113 ± 11                                                                          --  2899 ± 387                                                                         --                                     Antide                                                                              -44hr                                                                               3 50    90 ± 5                                                                            NS  2497 ± 155                                                                         NS                                     "     "    10 50     46 ± 12                                                                          <.001                                                                             1413 ± 230                                                                         <.01                                   "     "    30 50      5 ± 0.7                                                                         <.001                                                                             311 ± 34                                                                           <.001                                  VII-24                                                                              -24hr                                                                               3 50    19 ± 4                                                                            <.001                                                                              719 ± 123                                                                         <.001                                  "     "    10 50      3 ± 0.4                                                                         <.001                                                                             289 ± 30                                                                           <.001                                  "     "    30 50     0.3 ± 0.03                                                                       <.001                                                                             147 ± 10                                                                           <.001                                  IV-6(25897)                                                                         "     1 50     91 ± 19                                                                          NS  2020 ± 295                                                                         NS                                     "     "     3 50    42 ± 7                                                                            <.001                                                                             1298 ± 275                                                                         <.01                                   "     "    10 50    15 ± 3                                                                            <.001                                                                             624 ± 84                                                                           <.001                                  "     "    30 50    3.4 ± 2                                                                           <.001                                                                             273 ± 89                                                                           <.001                                  __________________________________________________________________________     *Mean of 6 ± SEM                                                             [Tyr.sup.5 ]-Antide                                                    

                                      TABLE VIII                                  __________________________________________________________________________    Duration of Action of Orally Administered Antide and Comparison with          [N--Ac--D-pCIPhe.sup.1,2,D--Trp.sup.3,D--Arg.sup.6,D--Ala.sup.10 ]-LHRH       (25).*                                                                        Time         0 Time                                                                             +2 hours                                                          of     Agonist                                                                            Serum      FSH                                                    adm.                                                                              Dose                                                                             Dose (sc)                                                                          LH ng/ml ±                                                                            ng/ml ±                                       Antagonist                                                                          hr  μg                                                                            ng   SEM    p value                                                                           SEM    p value                                   __________________________________________________________________________          --  -- --    3 ± 1                                                                            <.001                                                                             298 ± 20                                                                          <.001                                           --  --  5   21 ± 3                                                                            --   796 ± 102                                                                        --                                        Antide                                                                               -48                                                                              100                                                                               5     4 ± 0.8                                                                         <.001                                                                             481 ± 27                                                                          <.02                                      "      -48                                                                              300                                                                               5    8 ± 2                                                                            <.01                                                                              600 ± 72                                                                          NS                                        "      -24                                                                              100                                                                               5    9 ± 2                                                                            <.01                                                                              596 ±  50                                                                         NS                                        "      -24                                                                              300                                                                               5     6 ± 0.3                                                                         <.001                                                                             462 ± 54                                                                          <.02                                      "     -2   10                                                                               5   19 ± 4                                                                            NS  588 ± 70                                                                          NS                                        "     -2   30                                                                               5    6 ± 1                                                                            <.001                                                                             573 ± 67                                                                          NS                                        "     -2  100                                                                               5     1 ± 0.3                                                                         <.001                                                                             320 ± 48                                                                          <.01                                      "     -2  300                                                                               5    0.4 ± 0.4                                                                        <.001                                                                             327 ± 63                                                                          <.01                                      --    --  -- --    3 ± 1                                                                            <.001                                                                             298 ± 20                                                                          <.001                                     --    --  -- 10   44 ± 4                                                                            --  1488 ± 168                                                                        --                                        Antide                                                                               -48                                                                              100                                                                              10   18 ± 2                                                                            <.001                                                                              792 ± 110                                                                        <.01                                      "      -48                                                                              300                                                                              10   25 ± 3                                                                            <.01                                                                              1021 ± 202                                                                        NS                                        "      -24                                                                              100                                                                              10   24 ± 6                                                                            <.02                                                                              1008 ± 285                                                                        NS                                        "      -24                                                                              100                                                                              10   25 ± 3                                                                            <.01                                                                              1119 ± 71                                                                         NS                                        "     -2   10                                                                              10   51 ± 8                                                                            NS  1729 ± 243                                                                        NS                                        "     -2   30                                                                              10   22 ± 4                                                                            <.01                                                                              1051 ± 141                                                                        NS                                        "     -2  100                                                                              10    7 ± 3                                                                            <.001                                                                             548 ± 83                                                                          <.001                                     "     -2  300                                                                              10    0.5 ± .06                                                                        <.001                                                                             251 ± 24                                                                          <.001                                     VIII-25                                                                             -2   10                                                                              10    59 ± 11                                                                          NS  1794 ± 329                                                                        NS                                        "     -2   30                                                                              10   39 ± 6                                                                            NS  1470 ± 190                                                                        NS                                        "     -2  100                                                                              10   26 ± 7                                                                            <.05                                                                              1161 ± 277                                                                        NS                                        __________________________________________________________________________     *Kindly provided by Dr. David Coy                                               [D-Qal.sup.6)-LHRH                                                             Administered in water                                                 

                                      TABLE IX                                    __________________________________________________________________________    Oral Activity of Antide. Dependence on Vehicle.                               -2 hrs     0 Time                                                             Antagonist Agonist                                                                            +2 hrs                                                             Dose  Dose LH ng/nl ±                                                                            FSH ng/ml ±                                     Vehicle                                                                            μg oral                                                                          ng sc                                                                              SEM    p value                                                                           SEM     p value                                    __________________________________________________________________________    water                                                                              --    --   1.1 ± 0.1                                                                         <.001                                                                             243 ± 35                                                                           <.001                                      "    --    50   148 ± 9                                                                           --  3041 ± 238                                                                         --                                         "    100   50   44 ± 5                                                                            <.001                                                                             1372 ± 84                                                                          <.001                                      "    300   50   20 ± 4                                                                            <.001                                                                              936 ± 150                                                                         <.001                                      "     900* 50   6.3 ± 3                                                                           <.001                                                                             374 ± 80                                                                           <.001                                      corn oil                                                                           --    --   0.8 ± 0.6                                                                         <.001                                                                             138 ± 6                                                                            <.001                                      "    --    50   115 ± 8                                                                           --  2935 ± 133                                                                         --                                         "    100   50   72 ± 12                                                                           <.01                                                                              2148 ± 234                                                                         <.02                                       "    300   50   20 ± 4                                                                            <.001                                                                              792 ± 137                                                                         <.001                                      "    900   50   7 ± 2                                                                             <.001                                                                             599 ± 59                                                                           <.001                                      __________________________________________________________________________     Design: -2 hrs  Antagonist                                                    0 time  [D3-Qal.sup.6 ]-LHRH                                                  +2 hrs  Sacrifice                                                             26 day old female rates. Mean of 6 ± SEM                                   *Dilated 1:1 with 10 mM HOAC:Water (slightly cloudy) 0.1 ml orally, other     concentration diluted with water                                         

                  TABLE X                                                         ______________________________________                                        Oral Activity of Antide In the Antiovulatory Assay.*                          Oral        AOA                                                               Dose        % Inhibition                                                      μg       (# Ovulated/# Rats)                                               ______________________________________                                        --          O (6/6)                                                           300         18 (9/11)                                                         600         73 (3/11)                                                         1200        100 (0/11)                                                        ______________________________________                                         *in 10 mM acetic acid:water (1:1)                                        

                                      TABLE XI                                    __________________________________________________________________________    Oral Activity of Antide and Some Analogs.                                               0 Time                                                              -2 hrs    Agonist                                                                            +2 hrs                                                               Dose                                                                              Dose LH ng/ml ±                                                                            FSH ng/ml ±                                      Antagonist                                                                          μg oral                                                                        ng sc                                                                              SEM    p value                                                                           SEM     p value                                     __________________________________________________________________________          --  --    3.4 ± 2.2                                                                        <.001                                                                             271 ± 56                                                                           <.001                                             --  15     91 ± 4.6                                                                        --  2491 ± 146                                                                         --                                          Antide                                                                               30 15   75 ± 3                                                                            <.02                                                                              1718 ± 223                                                                         <.02                                        "     100 15   20 ± 4                                                                            <.001                                                                             738 ± 89                                                                           <.001                                       "     300 15    5 ± 1                                                                            <.001                                                                             472 ± 26                                                                           <.001                                       4      30 15   79 ± 9                                                                            NS  1831 ± 249                                                                         <.05                                        "     100 15   76 ± 6                                                                            NS  2175 ± 211                                                                         NS                                          "     300 15   51 ± 6                                                                            <.001                                                                             1404 ± 117                                                                         <.001                                       12     30 15   71 ± 9                                                                            NS  1965 ± 256                                                                         NS                                          "     100 15    54 ± 10                                                                          <.01                                                                              1031 ± 195                                                                         <.001                                       "     300 15     6 ± 1.1                                                                         <.001                                                                             514 ± 54                                                                           <.001                                       26*    30 15   75 ± 9                                                                            NS  2438 ± 207                                                                         NS                                          "     100 15   19 ± 3                                                                            <.001                                                                              845 ± 149                                                                         <.001                                       "     300 15     6 ± 1.4                                                                         <.001                                                                             431 ± 22                                                                           <.001                                       6      30 15    77 ± 12                                                                          NS  1761 ± 191                                                                         <.01                                        "     100 15    59 ± 12                                                                          <.05                                                                              1782 ± 388                                                                         NS                                          "     300 15    6.3 ± 1.4                                                                        <.001                                                                             467 ± 43                                                                           <.001                                             --  50   115 ± 15                                                                          --  2372 ± 126                                                                         --                                          Antide                                                                               30 50   93 ± 7                                                                            NS  2262 ± 55                                                                          NS                                          "     100 50   49 ± 7                                                                            <.001                                                                             1345 ± 199                                                                         <.001                                       "     300 50   19 ± 3                                                                            <.001                                                                             630 ± 40                                                                           <.001                                       "     900 50    5.3 ± 1.2                                                                        <.001                                                                             450 ± 48                                                                           <.001                                       __________________________________________________________________________     Design: -2 hrs  Antagonist                                                    0 Time  [D3-Qal.sup.6 ]-LHRH                                                  +2 hrs  Sacrifice                                                             26 day old female rats. Mean of 6 ± SEM                                    Vehicle  10 mM HOAC:Water (1:1) 0.1 ml                                        *[D--N.sup.ε -pyrazinylcarbonyllysyl.sup.6 ]-Antide              

                                      TABLE XII                                   __________________________________________________________________________    ORAL ACTIVITY OF ANTIDE                                                       At Various Time Schedules and Doses of a LH-RH Superagonist                   [NAcD2Nal.sup.1,DpClPhe.sup.2,D3Pal.sup.3,NicLys.sup.5,DNicLys.sup.6,ILys.    sup.8,DAla.sup.10 ]LHRH                                                       Antagonist                                                                    Time adm. Agonist*                                                                           LH           FSH                                               (oral)                                                                              Dosage                                                                            Dose (sc)                                                                          ng/ml ±                                                                          p value                                                                              ng/ml ±                                        hr    μg                                                                             0 TIME                                                                             SEM   +2 HOURS                                                                             SEM   p value                                     __________________________________________________________________________    --    --  --    3 ± 1                                                                           <.001  298 ± 20                                                                          <.001                                      --    --   5 ng                                                                              21 ± 3                                                                           --      796 ± 120                                                                       --                                           -48  100  5 ng                                                                                4 ± 0.8                                                                        <.001  481 ± 27                                                                         <.02                                         -48  300  5 ng                                                                               8 ± 2                                                                           <.01   600 ± 72                                                                         NS                                           -24  100  5 ng                                                                               9 ± 2                                                                           <.01   596 ± 50                                                                         NS                                           -24  300  5 ng                                                                                6 ± 0.3                                                                        <.001  462 ± 54                                                                         <.02                                        -2     10  5 ng                                                                              19 ± 4                                                                           NS     588 ± 70                                                                         NS                                          -2     30  5 ng                                                                               6 ± 1                                                                           <.001  573 ± 67                                                                         NS                                          -2    100  5 ng                                                                                1 ± 0.3                                                                        <.001  320 ± 48                                                                         <.01                                        -2    300  5 ng                                                                               0.4 ± 0.4                                                                       <.001  327 ± 63                                                                         <.01                                        --    --  --    3 ± 1                                                                           <.001  298 ± 20                                                                          <.001                                      --    --  10 ng                                                                              44 ± 4                                                                           --     1488 ± 168                                                                       --                                           -48  100 10 ng                                                                              18 ± 2                                                                           <.001   792 ± 110                                                                       <.01                                         -48  300 10 ng                                                                              25 ± 3                                                                           <.01   1021 ± 202                                                                       NS                                           -24  100 10 ng                                                                              24 ± 6                                                                           <.02   1008 ± 285                                                                       NS                                           -24  300 10 ng                                                                              25 ± 3                                                                           <.01   1119 ± 71                                                                        NS                                          -2     10 10 ng                                                                              51 ± 8                                                                           NS     1729 ± 243                                                                       NS                                          -2     30 10 ng                                                                              22 ± 4                                                                           <.01   1051 ± 141                                                                       NS                                          -2    100 10 ng                                                                               7 ± 3                                                                           <.001  548 ± 83                                                                          <.001                                      -2    300 10 ng                                                                               0.5 ± .06                                                                       <.001  251 ± 24                                                                          <.001                                      -2     10**                                                                             10 ng                                                                               59 ± 11                                                                         NS     1794 ± 329                                                                       NS                                          -2     30 10 ng                                                                              39 ± 6                                                                           NS     1470 ± 190                                                                       NS                                          -2    100 10 ng                                                                              26 ± 7                                                                           <.05   1161 ± 277                                                                       NS                                          __________________________________________________________________________     *24270 [D3Qal.sup.6 ]-LHRH                                                    **AH-195-3 NAcDpClPhe.sup.1,2,DTrp.sup.3,DAla.sup.10 -LHRH (Dr. David Coy     mean of 6 ± SEM                                                       

                                      TABLE XIII                                  __________________________________________________________________________    Effect of Antide in the                                                       Dispersed Pituitary Cell Culture Assay                                             Dose                                                                             LHRH                                                                              RLH ng/ml ±  FSH ng/ml ±                                    Peptide                                                                            nM nM  SEM     p value                                                                           IDR.sub.50                                                                        SEM     p value                                                                           IDR.sub.50                            __________________________________________________________________________    Control                                                                            -- --    10 ± 0.4                                                                         --      196 ± 23                                                                           --                                        LHRH -- 0.1 40 ± 7                                                                             <.05    221 ± 18                                                                           NS                                             -- 0.3 80 ± 1                                                                             <.001   562 ± 48                                                                           ≈.02                                   -- 1.0 118 ±                                                                              NA      802 ±                                                                              NA                                             -- 3.0 150 ± 1                                                                            <.001                                                                             --   646 ± 123                                                                         NS                                             -- 10.0                                                                              141 ± 4                                                                            <.001   602 ± 26                                                                           <.01                                           -- 30.0                                                                              152 ± 7                                                                            <.01    557 ± 15                                                                           <.01                                      139-95-                                                                            0.01                                                                             3.0 118 ± 11                                                                             NS*                                                                             0.26:1                                                                            546 ± 93                                                                            NS*                                                                              0.52:1                                20   0.03                                                                             3.0 117 ± 10                                                                           NS      499 ± 26                                                                           NS                                              0.1                                                                             3.0 116 ± 7                                                                            <.05    472 ± 59                                                                           NS                                              0.3                                                                             3.0 107 ± 11                                                                           NS      617 ± 73                                                                           NS                                              1.0                                                                             3.0 80 ± 2                                                                             <.001   481 ± 17                                                                           NS                                              3.0                                                                             3.0 34 ± 2                                                                             <.001   233 ± 38                                                                           NS                                             10.0                                                                             3.0 11 ± 1                                                                             <.001   165 ± 21                                                                           NS                                        __________________________________________________________________________     *p values vs 3 nM of LHRH                                                     139-95-20                                                                     [NAcD2Nal.sup.1,DpCIPhe.sup.2,D3Pal.sup.3,NicLys.sup.5,DNicLys.sup.6,ILys    sup.8,DAla.sup.10 ]LHRH                                                   

                                      TABLE XIV                                   __________________________________________________________________________    LHRH analogs with 50% or more AOA at 0.25 ug                                                                                   AOA/                         IBR# Sequence                                    0.25                                                                              Wheal                                                                               EDea               __________________________________________________________________________    25897                                                                              N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, PicLys, c-D--PzACAla,Leu,ILys,Pro         ,D--Ala--NH.sub.2                           73  122.8                                                                               28.sup.50                                                                     ±                                                                          7.5                26325                                                                              N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, PicLys, c-D--PzACAla,Val,ILys,Pro         ,D--Ala--NH.sub.2                           73  127.8 ± 4.9           26180                                                                              N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, c-PzACAla, D-PicLys                       Leu,ILys,Pro,D--Ala--NH.sub.2               67   99.5 ± 4.5           26326                                                                              N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, PicLys, c-D--PzACAla                      Leu,ILys,Pro,D--Ala--NH.sub.2               57  115.5 ± 2.4           26181                                                                              N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, PicLys c-D--PzACAla,Leu, IOrn             Pro,D--Ala--NH.sub.2                        50  101.6 ± 2.2           *26933                                                                             N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, PicLys, D--PicLys                         Leu,ILys,Pro,D--Ala--NH.sub.2               50  113.0 ± 0             26346                                                                              N--Ac--D-2-Nal,DpClPhe,D-3-Pal,Ser, PicLys, D--PicLys Abu Arg                 Pro,D--Ala--NH.sub.2                        50  113.2                    __________________________________________________________________________                                                         ± 5.4                  *Claimed in original                                                     

REFERENCES

1. Ljungqvist, A. , Feng, D. -M. , Tang, P. -F. L. , Kubota, M.,Okamoto, T., Zhang, Y., Bowers, C. Y., Hook, W. A. & Folkers K. (1987)Biochem. Biophys. Res. Commun. 148 (2), 849-856.

2. Karten, M. D. & Rivier, J. E. (1986) Endocr. Rev. 7, 44-56.

3. Hook, W. A., Karten, M. & Siraganian, R. P. (1985) Fed. Proc. Fed.Am. Soc. Exptl. Biol. 44, 1323.

4. Karten, M. D., Hook, W. A., Siraganian, R. P., Coy, D. H., Folkers,K., Rivier, J. E. & Roeske, R. W. (1987) in LHRH and its Analogs;Contraceptive and Therapeutic Applications Part 2, eds. Vickery, B. H. &Nestor, J. J. Jr., (MTP press Ltd., Lancaster, England) PP. 179-190.

5. Rivier, J. E., Porter, J., Rivier, C. L., Perrin, M., Corrigan, A.,Hook, W. A., Siraganian, R. P. & Vale, W. W. (1986) J. Med. Chem. 29,1846-1851.

6. Roeske, R. W., Chaturvedi, N. C., Hrinyo-Pavlina, T., & Kowalczuk, M.(1987) in LHRH and its Analogs; Contraceptive and TherapeuticApplications Part 2, eds. Vickery, B. H. & Nestor, J. J., Jr., (MTPpress Ltd. , Lancaster, England) pp. 17-24.

7. Hocart, S. J., Nekola, M. V. & Coy, D. H. (1987) J. Med Chem. 30,739-743.

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9. Bajusz, S., Kovacs, M., Gazdag, M., Bokser, L., Karashima, T.,Csernus, V. J., Janaky, T., Guoth, J. & Schally, A. V. (1988) Proc.Natl. Acad. Sci. USA 85, 1637-1641.

10. Rivier, J., Kupryszewski, G., Varga, J., Porter, J., Rivier, C.,Perrin, M., Hagler, A., Struthers, S., corrigan, A. & Vale, W. (1988) J.Med. Chem. 31, 677-682.

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Changes may be made in the particular amino acid or derivatives andtheir assembly described herein or in the steps or the sequence of stepsof the method described herein without departing from the concept andscope of the invention as defined in the following claims.

What is claimed is:
 1. An LHRH antagonist having low histamine releasingactivity comprising a decapeptide of the formula AA¹ -D-pClPhe² -D-3Pal³-Ser⁴ -AA⁵ -AA⁶ -AA⁷ -AA⁸ -Pro⁹ -D-Ala¹⁰ -NH₂, whereinAA¹ isN-Ac-D-2Nal, N-Ac-D-pClPhe or N-Ac-D-Cl₂ Phe; AA⁵ is Tyr, NicLys,PicLys, MNicLys, MPicLys, INicLys, DMGLys, PzcLys, or c-PzACAla; AA⁶ isD-NicLys, D-PicLys, D-MNicLys, D-MPicLys, D-INicLys, D-BzLys, D-PzcLys,D-PzACAla, D-NACAla, or D-PACAla; AA⁷ is Leu, Aile, Nle, Val, NVal, Abu,or Ala; and AA⁸ is ILys or IOrn.
 2. A decapeptide according to claim 1wherein AA¹ is N--Ac--D--2Nal.
 3. A decapeptide according to claim 2wherein AA⁷ is Leu.
 4. A decapeptide according to claims 1, 2 or 3wherein AA⁵ is Tyr.
 5. A decapeptide according to claim 4 wherein AA⁶ isD--NicLys.
 6. A decapeptide according to claim 1, 2 or 3 wherein AA⁵ isNicLys, PicLys, MNicLys, MPicLys, INicLys, DMGLys, or PzcLys; and AA⁶ isD-NicLys, D-PicLys, D-MNicLys, D-MPicLys, D-INicLys, D-BzLys, orD-PzcLys.
 7. A decapeptide according to claim 1, 2 or 3 wherein AA⁵ isNicLys or PicLys and AA⁶ is D-PzACAla, D-NACAla, or D-PACAla.
 8. Adecapeptide according to claim 2 wherein AA⁵ is DMGLys, AA⁶ is D-NicLys,AA⁷ is Leu, and AA⁸ is ILys.
 9. A decapeptide according to claim 2wherein AA⁵ is PicLys, AA⁶ is D-NicLys, AA⁷ is Leu, and AA⁸ is ILys. 10.A decapeptide according to claim 2 wherein AA⁵ is INicLys, AA⁶ isD-INicLys , AA⁷ is Leu, and AA⁸ is ILys.
 11. A decapeptide according toclaim 2 wherein AA⁵ is NicLys, AA⁶ is D-BzLys, AA⁷ is Leu, and AA⁸ isILys.
 12. A decapeptide according to claim 2 wherein AA⁵ is NicLys, AA⁶is D-PicLys, AA⁷ is Leu, and AA⁸ is ILys.
 13. A decapeptide according toclaim 2 wherein AA⁵ is NicLys, AA⁶ is D-PzcLys, AA⁷ is Leu, and AA⁸ isILys.
 14. A decapeptide according to claim 2 wherein AA⁵ is NicLys, AA⁶is D-PzcLys, AA⁷ is Leu, and AA⁸ is IOrn.
 15. A decapeptide according toclaim 2 wherein AA⁵ is NicLys, AA⁶ is D-PzACAla, AA⁷ is Leu, and AA⁸ isILys.
 16. A decapeptide according to claim 2 wherein AA⁵ is NicLys, AA⁶is D-NACAla, AA⁷ is Leu, and AA⁸ is ILys.
 17. A decapeptide according toclaim 2 wherein AA⁵ is PicLys, AA⁶ is D-PicLys, AA⁷ is Val, and AA⁸ isILys.
 18. A decapeptide according to claim 2 wherein AA⁵ is PicLys, AA⁶is D-PicLys, AA⁷ is Val, and AA⁸ is IOrn.
 19. A decapeptide according toclaim 2 wherein AA⁵ is PicLys, AA⁶ is D-PicLys, AA⁷ is Aile, and AA⁸ isILys.
 20. A decapeptide according to claim 2 wherein AA⁵ is PicLys, AA⁶is D-PicLys, AA⁷ is Aile, and AA⁸ is IOrn.
 21. A decapeptide accordingto claim 27 wherein AA⁵ is PicLys, AA⁶ is D-PicLys, AA⁷ is Abu, and AA⁸is ILys.
 22. A decapeptide according to claim 2 wherein AA⁵ is PicLys,AA⁶ is D-PicLys, AA⁷ is Abu, and AA⁸ is IOrn.
 23. A decapeptideaccording to claim 2 wherein AA⁵ is PicLys, AA⁶ is D-PicLys, AA⁷ is Nle,and AA⁸ is ILys.
 24. A decapeptide according to claim 2 wherein AA⁵ isPicLys, AA⁶ is D-PicLys, AA⁷ is NVal, and AA⁸ is ILys.
 25. A decapeptideaccording to claim 2 wherein AA⁵ is PicLys, AA⁶ is D-PicLys, AA⁷ is Ala,and AA⁸ is ILys.
 26. A decapeptide according to claim 2 wherein AA⁵ isPicLys, AA⁶ is c-D-PzACAla, AA⁷ is Leu, and AA⁸ is ILys.
 27. Adecapeptide according to claim 2 wherein AA⁵ is PicLys, AA⁶ isc-D-PzACAla, AA⁷ is Leu, and AA⁸ is IOrn.
 28. A decapeptide according toclaim 2 wherein AA⁵ is PicLys, AA⁶ is c-D-PzACAla, AA⁷ is Val, and AA⁸is ILys.
 29. A decapeptide according to claim 2 wherein AA⁵ is PicLys,AA⁶ is D-PACAla, AA⁷ is Leu, and AA⁸ is ILys.
 30. A decapeptideaccording to claim 2 wherein AA⁵ is PicLys, AA⁶ is t-D-PzACAla, AA⁷ isLeu, and AA⁸ is ILys.
 31. A decapeptide according to claim 2 wherein AA⁵is c-PzACAla, AA⁶ is D-PicLys, AA⁷ is Leu, and AA⁸ is ILys.
 32. Adecapeptide according to claim 2 wherein AA⁵ is c-PzACAla, AA⁶ isc-D-PzACAla, AA⁷ is Leu, and AA⁸ is ILys.
 33. A decapeptide according toclaim 2 wherein AA⁵ is c-PzACAla, AA⁶ is D-PicLys, AA⁷ is Val, and AA⁸is ILys.
 34. A decapeptide according to claim 27 wherein AA⁵ is MPicLys,AA⁶ is D-MPicLys, AA⁷ is Leu, and AA⁸ is ILys.
 35. A decapeptideaccording to claim 2 wherein AA⁵ is DMGLys, AA⁶ is D-BzLys, AA⁷ is Leu,and AA⁸ is ILys.
 36. A decapeptide according to claim 2 wherein AA⁵ isTyr, AA⁶ is D-NicLys, AA⁷ is Leu, and AA⁸ is ILys.
 37. A decapeptideaccording to claim 2 wherein AA⁵ is Tyr, AA⁶ is D-NicLys, AA⁷ is Leu,and AA⁸ is IOrn.
 38. A decapeptide according to claim 2 wherein AA⁵ isTyr, AA⁶ is c-D-PzACAla, AA⁷ is Leu, and AA⁸ is ILys.
 39. A decapeptideaccording to claim 1 wherein AA¹ is N-Ac-D-pClPhe, AA⁵ is Tyr, AA⁶ isD-NicLys, AA⁷ is Leu, and AA⁸ is ILys.
 40. A decapeptide according toclaim 1 wherein AA¹ is N-Ac-D-Cl₂ Phe, AA⁵ is Tyr, AA⁶ is D-NicLys, AA⁷is Leu, and AA⁸ is ILys.
 41. A decapeptide according to claim 1 which atdosage of 1 μg has an antiovulatory activity of about 90% or more, whenmeasured according to the test of Humphties et al. (J. Med. Chem.21:120-23 (1978)).
 42. A decapeptide according to claim 41 having ahistamine release corresponding to an ED₅₀ value of about 86/μg/ml ormore, when measured according to the test of Karten et al. (LHRH and itsAnalogs: Contraceptive and Therapeutic Applications Part 2, pp. 179-90(1987)).